Methods and compositions relating to adenosine receptors

ABSTRACT

Provided herein are methods and compositions relating to adenosine A2A receptor libraries having nucleic acids encoding for a scaffold comprising an adenosine A2A binding domain. adenosine A2A receptor libraries described herein encode for immunoglobulins such as antibodies.

CROSS-REFERENCE

This application claims the benefit of U.S. Patent Application No.63/140,201, filed Jan. 21, 2021, U.S. Patent Application No. 63/209,892,filed Jun. 11, 2021, and U.S. Patent Application No. 63/244,976, filedSep. 16, 2021, the contents of each of which is entirely incorporated byreference herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 3, 2022, isnamed 44854-821_201_SL.txt and is 343,338 bytes in size.

BACKGROUND

G protein-coupled receptors (GPCRs) such as adenosine receptors areimplicated in a wide variety of diseases. Raising antibodies to GPCRshas been difficult due to problems in obtaining suitable antigen becauseGPCRs are often expressed at low levels in cells and are very unstablewhen purified. Thus, there is a need for improved agents for therapeuticintervention which target adenosine receptors.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF SUMMARY

Provided herein are compositions and methods for activating T cells.

Provided herein are methods for activating T cells, comprisingadministering an antibody or antibody fragment comprising a sequence atleast about 90% identical to a sequence set forth in SEQ ID NOs: 6-717.Further provided herein are methods for activating T cells, wherein theantibody or antibody fragment comprises an amino acid sequence at leastabout 95% identical to that set forth in any one of SEQ ID NOs: 35-44.Further provided herein are methods for activating T cells, wherein theantibody or antibody fragment comprises an amino acid sequence as setforth in any one of SEQ ID NOs: 35-44. Further provided herein aremethods for activating T cells, wherein the antibody is a monoclonalantibody, a polyclonal antibody, a bi-specific antibody, a multispecificantibody, a grafted antibody, a human antibody, a humanized antibody, asynthetic antibody, a chimeric antibody, a camelized antibody, asingle-chain Fvs (scFv), a single chain antibody, a Fab fragment, aF(ab′)2 fragment, a Fd fragment, a Fv fragment, a single-domainantibody, an isolated complementarily determining region (CDR), adiabody, a fragment comprised of only a single monomeric variabledomain, disulfide-linked Fvs (sdFv), an intrabody, an anti-idiotypic(anti-Id) antibody, or ab antigen-binding fragments thereof. Furtherprovided herein are methods for activating T cells, wherein the antibodyor antibody fragment binds to adenosine 2A receptor with a K_(D) of lessthan about 75 nM. Further provided herein are methods for activating Tcells, wherein the antibody or antibody fragment binds to adenosine 2Areceptor with a K_(D) of less than about 50 nM. Further provided hereinare methods for activating T cells, wherein the antibody or antibodyfragment binds to adenosine 2A receptor with a K_(D) of less than about25 nM. Further provided herein are methods for activating T cells,wherein the antibody or antibody fragment binds to adenosine 2A receptorwith a K_(D) of less than about 10 nM. Further provided herein aremethods for activating T cells, wherein the antibody or antibodyfragment comprises an IC₅₀ of less than about 20 nM in a T cellactivation assay. Further provided herein are methods for activating Tcells, wherein the antibody or antibody fragment comprises an IC₅₀ ofless than about 10 nM in a T cell activation assay. Further providedherein are methods for activating T cells, wherein the antibody orantibody fragment comprises an IC₅₀ of less than about 7.5 nM in a Tcell activation assay. Further provided herein are methods foractivating T cells, wherein the antibody or antibody fragment comprisesan IC₅₀ of less than about 5 nM in a T cell activation assay.

Provided herein are antibodies or antibody fragments comprising asequence at least about 90% identical to a sequence set forth in SEQ IDNOs: 6-717. Further provided herein are antibodies or antibody fragmentswherein the antibody or antibody fragment comprises an amino acidsequence at least about 95% identical to that set forth in any one ofSEQ ID NOs: 35-44. Further provided herein are antibodies or antibodyfragments wherein the antibody or antibody fragment comprises an aminoacid sequence as set forth in any one of SEQ ID NOs: 35-44. Furtherprovided herein are antibodies or antibody fragments wherein theantibody is a monoclonal antibody, a polyclonal antibody, a bi-specificantibody, a multispecific antibody, a grafted antibody, a humanantibody, a humanized antibody, a synthetic antibody, a chimericantibody, a camelized antibody, a single-chain Fvs (scFv), a singlechain antibody, a Fab fragment, a F(ab′)2 fragment, a Fd fragment, a Fvfragment, a single-domain antibody, an isolated complementarilydetermining region (CDR), a diabody, a fragment comprised of only asingle monomeric variable domain, disulfide-linked Fvs (sdFv), anintrabody, an anti-idiotypic (anti-Id) antibody, or ab antigen-bindingfragments thereof. Further provided herein are antibodies or antibodyfragments wherein the antibody or antibody fragment binds to adenosine2A receptor with a K_(D) of less than about 75 nM. Further providedherein are antibodies or antibody fragments wherein the antibody orantibody fragment binds to adenosine 2A receptor with a KD of less thanabout 50 nM. Further provided herein are antibodies or antibodyfragments wherein the antibody or antibody fragment binds to adenosine2A receptor with a KD of less than about 25 nM. Further provided hereinare antibodies or antibody fragments wherein the antibody or antibodyfragment binds to adenosine 2A receptor with a KD of less than about 10nM. Further provided herein are antibodies or antibody fragments whereinthe antibody or antibody fragment comprises an IC50 of less than about20 nM in a T cell activation assay. Further provided herein areantibodies or antibody fragments wherein the antibody or antibodyfragment comprises an IC50 of less than about 10 nM in a T cellactivation assay. Further provided herein are antibodies or antibodyfragments wherein the antibody or antibody fragment comprises an IC50 ofless than about 7.5 nM in a T cell activation assay. Further providedherein are antibodies or antibody fragments wherein the antibody orantibody fragment comprises an IC50 of less than about 5 nM in a T cellactivation assay.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts a first schematic of an immunoglobulin scaffold.

FIG. 1B depicts a second schematic of an immunoglobulin scaffold.

FIG. 2 depicts a schematic of a motif for placement in a scaffold.

FIG. 3 presents a diagram of steps demonstrating an exemplary processworkflow for gene synthesis as disclosed herein.

FIG. 4 illustrates an example of a computer system.

FIG. 5 is a block diagram illustrating an architecture of a computersystem.

FIG. 6 is a diagram demonstrating a network configured to incorporate aplurality of computer systems, a plurality of cell phones and personaldata assistants, and Network Attached Storage (NAS).

FIG. 7 is a block diagram of a multiprocessor computer system using ashared virtual address memory space.

FIG. 8A depicts a schematic of an immunoglobulin scaffold comprising aVH domain attached to a VL domain using a linker.

FIG. 8B depicts a schematic of a full-domain architecture of animmunoglobulin scaffold comprising a VH domain attached to a VL domainusing a linker, a leader sequence, and pIII sequence.

FIG. 8C depicts a schematic of four framework elements (FW1, FW2, FW3,FW4) and the variable 3 CDR (L1, L2, L3) elements for a VL or VH domain.

FIG. 9A depicts a structure of Glucagon-like peptide 1 (GLP-1, boxed) incomplex with GLP-1 receptor (GLP-1R), PDB entry 5VAI.

FIG. 9B depicts a crystal structure of CXCR4 chemokine receptor incomplex with a cyclic peptide antagonist CVX15 (boxed), PDB entry 3OR0.

FIG. 9C depicts a crystal structure of human smoothened with thetransmembrane domain and extracellular domain (ECD) (boxed), PDB entry5L7D. The ECD contacts the TMD through extracellular loop 3 (ECL3).

FIG. 9D depicts a structure of GLP-1R in complex with a Fab (boxed), PDBentry 6LN2.

FIG. 9E depicts a crystal structure of CXCR4 in complex with a viralchemokine antagonist Viral macrophage inflammatory protein 2 (vMIP-II,boxed), PDB entry 4RWS.

FIG. 10 depicts a schema of the GPCR focused library design. Twogermline heavy chain VH1-69 and VH3-30; 4 germline light chain IGKV1-39and IGKV3-15, and IGLV1-51 and IGLV2-14.

FIG. 11 depicts a graph of HCDR3 length distribution in the GPCR-focusedlibrary compared to the HCDR3 length distribution in B-cell populationsfrom three healthy adult donors. In total, 2,444,718 unique VH sequencesfrom the GPCR library and 2,481,511 unique VH sequences from humanB-cell repertoire were analyzed to generate the length distributionplot. The y-axis is labeled frequency from 0.000 to 0.1400 at 0.0200unit intervals; the x-axis is length and labeled 0 to 57 at 3 amino acidintervals.

FIG. 12 depicts the clone, ELISA value, Library, ProA value, and K_(D)value for VHH-Fc.

FIG. 13 depicts a schema of design of phage-displayed hyperimmunelibraries generated herein.

FIGS. 14A-14B depict graphs of a dose curve (FIG. 14A) and FACS analysis(FIG. 14B) of A2AR-90-007.

FIG. 15A depicts a schema of heavy chain IGHV3-23 design. FIG. 15Adiscloses SEQ ID NOS 726-731, respectively, in order of appearance.

FIG. 15B depicts a schema of heavy chain IGHV1-69 design. FIG. 15Bdiscloses SEQ ID NOS 732-737, respectively, in order of appearance.

FIG. 15C depicts a schema of light chains IGKV 2-28 and IGLV 1-51design. FIG. 15C discloses SEQ ID NOS 738-743, respectively, in order ofappearance.

FIG. 15D depicts a schema of the theoretical diversity and finaldiversity of a GPCR library.

FIGS. 16A-16O depict flow cytometry data using variant A2A receptorimmunoglobulins A2A90 (FIG. 16A), A2A91 (FIG. 16B), A2A92 (FIG. 16C),A2A93 (FIG. 16D), A2A94 (FIG. 16E), A2A1 (FIG. 16F), A2A95 (FIG. 16G),A2A2 (FIG. 16H), A2A3 (FIG. 16I), A2A4 (FIG. 16J), A2A5 (FIG. 16K), A2A6(FIG. 16L), A2A96 (FIG. 16M), A2A7 (FIG. 16N), and control (FIG. 16O).

FIGS. 17A-17H depict graphs of binding curves using variant A2A receptorimmunoglobulins A2A-94 (FIG. 17A), A2A1 (FIG. 17B), A2A3 (FIG. 17C),A2A4 (FIG. 17D) A2A5 (FIG. 17E), A2A6 (FIG. 17F), A2A7 (FIG. 17G), andcontrol (FIG. 17H). Binding curves are plotted with IgG concentrationvs. MFI (mean fluorescence intensity).

FIGS. 18A-18O depict graphs of binding curves using variants from amouse immune library: A2A97 (FIG. 18A), A2A98 (FIG. 18B), A2A99 (FIG.18C), A2A100 (FIG. 18D), A2A101 (FIG. 18E), A2A102 (FIG. 18F), A2A103(FIG. 18G), A2A104 (FIG. 18H), A2A9 (FIG. 18I), A2A10 (FIG. 18J), A2A11(FIG. 18K), A2A12 (FIG. 18L), A2A13 (FIG. 18M), A2A14 (FIG. 18N), andusing a control (FIG. 18O).

FIGS. 19A-19G depict graph of cell binding with adenosine A2aRmonoclonal (MAB9497) and selected variants: A2A-9 (FIG. 19A), A2A10(FIG. 19B), A2A11 (FIG. 19C), A2A12 (FIG. 19D) A2A13 (FIG. 19E), A2A15(FIG. 19F), and control (FIG. 19G). Binding curves are plotted with IgGconcentration vs. MFI (mean fluorescence intensity).

FIGS. 20A-20G depict graphs of cell binding in a titration assay from100 nM. Graphs depict cell binding of a synthetic library to A2a protein(FIG. 20A), synthetic library to A2a protein+ZM241385 (FIG. 20B),humanized synthetic library to A2a protein (FIG. 20C), humanizedsynthetic library to A2a protein+ZM241385 (FIG. 20D), immune library toA2a protein (FIG. 20E), immune library to A2a protein+ZM241385 (FIG.20F), and mouse immune library to A2a protein (FIG. 20G).

FIG. 21 depicts data from an agonist dose-response assay measured usinga cAMP assay.

FIG. 22 depicts data from an antagonist dose-response assay measuredusing a cAMP assay.

FIG. 23 depicts results from a cAMP antagonist titration assay.

FIG. 24 depicts data from variant A2A-1 and A2A-9 from a cAMP assay.

FIG. 25 depicts data for variant A2A9 using a cAMP assay.

FIG. 26 depicts data for variant A2A9 using a cAMP antagonist titrationassay.

FIG. 27A depicts data for variant A2A receptor immunoglobulins in anantagonistic cAMP assay. FIG. 27B depicts data for additional variantA2A receptor immunoglobulins in an antagonistic cAMP assay. FIG. 27Cdepicts data for additional variant A2A receptor immunoglobulins in anantagonistic cAMP assay.

FIG. 28A depicts data for variant A2A receptor immunoglobulins in anallosteric cAMP assay. FIG. 28B depicts data for additional variant A2Areceptor immunoglobulins in an allosteric cAMP assay. FIG. 28C depictsdata for additional variant A2A receptor immunoglobulins in anallosteric cAMP assay.

FIG. 29A depicts data for variant A2A receptor immunoglobulins in anantagonistic cAMP assay. FIG. 29B depicts data for additional variantA2A receptor immunoglobulins in an antagonistic cAMP assay. FIG. 29Cdepicts data for additional variant A2A receptor immunoglobulins in anantagonistic cAMP assay.

FIG. 30A depicts data for variant A2A receptor immunoglobulins in anantagonistic cAMP assay. FIG. 30B depicts data for additional variantA2A receptor immunoglobulins in an antagonistic cAMP assay. FIG. 30Cdepicts data for additional variant A2A receptor immunoglobulins in anantagonistic cAMP assay.

FIGS. 31A-31C depict affinity data (FIG. 31A), additional affinity data(FIG. 31B), and specificity data (FIG. 31C) for variant A2A-77.

FIG. 31D depicts A2A-77 binding to cynomolgus PBMCs.

FIG. 32A depicts T cell activation for variants A2A-81, A2A-51, A2A-53,A2A-77, A2A-31, A2A-24, A2A-78, A2A-74, A2A-75, A2A-52, and A2A-36. FIG.32B depicts T cell activation for variants A2A-81, A2A-51, A2A-53,A2A-77, A2A-31, and A2A-78.

FIG. 32C depicts T cell activation data for variant A2A-77.

FIGS. 32D-32H depict T cell activation data for variants A2A-81, A2A-51,A2A-77, and A2A-28.

FIG. 33A depicts the result of a cell binding assay for variants A2A-77and A2A-81.

FIG. 33B depicts the result of an A2A antagonistic cAMP assay forvariants A2A-77 and A2A-81.

FIG. 33C depicts specificity data for variants A2A-77 and A2A-81, aswell as a control A2a.

FIG. 33D depicts T cell activation data for variants A2A-77 and A2A-81.

FIGS. 34A-34B depicts the mean tumor volume over time (FIGS. 34A and34C) and the relative tumor volume over time (FIGS. 34B and 34D) of micetreated with variants A2A-77 and A2A-81.

FIG. 34E depict the experimental schema for combination treatments.

FIGS. 34F-34K depict data from the colon cancer model.

FIGS. 35A-35M depict the proportion amount of cells detected in mice ineach of the four treatment groups. FIG. 35A depicts the number of TILCD45+ cells as a percent of all live cells detected. FIGS. 35B-35Gdepict the number of total T-cells (FIG. 35B), CD4+ cells (FIG. 35C),CD8+ cells (FIG. 35D), regulatory T-cells (Tregs, FIG. 35E), M1 tumorassociated macrophages (TAM, FIG. 35F) and M2 TAM (FIG. 35G). FIG. 35Hdepicts the number of TIL CD45+ cells as a percent of all live cellsdetected. FIGS. 35I-35J depict the number of total T-cells (FIG. 35I),CD4+ cells (FIG. 35J), CD8+ cells (FIG. 35K), regulatory T-cells (Tregs,FIG. 35L), and M1 tumor associated macrophages (TAM, FIG. 35M).

FIGS. 36A-36C depicts a cell profile of lysed whole blood in interimversus terminal sample. The percent of CD45+ cells is depicted as apercent of live cells (FIG. 36A). The amount of CD3+ (FIG. 36B) and CD3−(FIG. 36C) cells is depicted as a percent of CD45+ cells.

FIGS. 37A-37G depict the proportion amount of cells detected in mice ineach of the four treatment groups in interim lysed whole blood samples.FIG. 37A depicts the number of TIL CD45+ cells as a percent of all livecells detected. FIGS. 37B-37G depict the number of total T-cells (FIG.37B), CD4+ cells (FIG. 37C), CD8+ cells (FIG. 37D), regulatory T-cells(Tregs, FIG. 37E), M1 tumor associated macrophages (TAM, FIG. 37F) andM2 TAM (FIG. 37G).

FIGS. 38A-38G depict the proportion amount of cells detected in mice ineach of the four treatment groups in interim lysed whole blood samples.FIG. 38A depicts the number of TIL CD45+ cells as a percent of all livecells detected. FIGS. 38B-38G depict the number of total T-cells (FIG.38B), CD4+ cells (FIG. 38C), CD8+ cells (FIG. 38D), regulatory T-cells(Tregs, FIG. 38E), M1 tumor associated macrophages (TAM, FIG. 38F) andM2 TAM (FIG. 38G).

FIG. 39 depicts cytokine level in peripheral blood after T cellactivation.

FIGS. 40A-40G depicts levels of interferon gamma (FIG. 40A), interleukin2 (FIG. 40B), interleukin 4 (FIG. 40C), interleukin 6 (FIG. 40D),interleukin 8 (FIG. 40E), interleukin 10 (FIG. 40F), and TNF alpha (FIG.40G) detected in terminal blood samples.

FIGS. 41A-41C depicts a cell profile of lysed whole blood in interimversus terminal sample. The percent of CD45+ cells is depicted as apercent of live cells (FIG. 41A). The amount of CD3+ (FIG. 41B) and CD3−(FIG. 41C) cells is depicted as a percent of CD45+ cells.

FIGS. 42A-42G depict the proportion amount of cells detected in mice ineach of the four treatment groups in interim lysed whole blood samples.FIG. 42A depicts the number of TIL CD45+ cells as a percent of all livecells detected. FIGS. 42B-42G depict the number of total T-cells (FIG.42B), CD4+ cells (FIG. 42C), CD8+ cells (FIG. 42D), regulatory T-cells(Tregs, FIG. 42E), M1 tumor associated macrophages (TAM, FIG. 42F) andM2 TAM (FIG. 42G).

FIGS. 43A-43G depict the proportion amount of cells detected in mice ineach of the four treatment groups in terminal lysed whole blood samples.FIG. 43A depicts the number of TIL CD45+ cells as a percent of all livecells detected. FIGS. 43B-43G depict the number of total T-cells (FIG.43B), CD4+ cells (FIG. 43C), CD8+ cells (FIG. 43D), regulatory T-cells(Tregs, FIG. 43E), M1 tumor associated macrophages (TAM, FIG. 43F) andM2 TAM (FIG. 43G).

FIGS. 44A-44G depicts levels of interferon gamma (FIG. 44A), interleukin2 (FIG. 44B), interleukin 4 (FIG. 44C), interleukin 6 (FIG. 44D),interleukin 8 (FIG. 44E), interleukin 10 (FIG. 44F), and TNF alpha (FIG.44G) detected in terminal blood samples.

FIG. 45 depicts hA2b cross binder activity in HEK293T cells.

FIG. 46 depicts the functional cAMP assay used to test the activity ofthe A2b antibodies.

FIGS. 47A-47D depict the results of the A2b functional cAMP assays.

FIGS. 48A-48E demonstrate primary T cell activation assays (cytokinerelease) in response to reformatted antibodies (IgG1 or IgG4).

FIGS. 49A-49L depict the proportion amount of cells detected in mice ineach of the treatment groups in interim lysed whole blood samples. FIG.49A depicts the number of LWB CD45+ cells as a percent of all live cellsdetected. FIGS. 49B-49L depict the number of total CD3+ cells (FIG.49B), CD8+ cells (FIG. 49C), CD4+ cells (FIG. 49D), CD3− non-T-cells(FIG. 49E), Tregs cells (FIG. 49F), proliferative T-cells (FIG. 49G),proliferative Tregs cells (FIG. 49H), CD11b+ cells (FIG. 49I), CD11c+cells (FIG. 49J), M1 macrophages (FIG. 49K), and M2 macrophages (FIG.49L).

FIGS. 50A-50L depict the proportion amount of cells detected in mice ineach of the treatment groups in terminal lysed whole blood samples. FIG.50A depicts the number of LWB CD45+ cells as a percent of all live cellsdetected. FIGS. 50B-50L depict the number of total CD3+ cells (FIG.50B), CD4+ cells (FIG. 50C), CD8+ cells (FIG. 50D), CD3− non-T-cells(FIG. 50E), Tregs cells (FIG. 50F), proliferative T-cells (FIG. 50G),proliferative Tregs cells (FIG. 50H), CD11b+ cells (FIG. 50I), CD11c+cells (FIG. 50J), M1 macrophages (FIG. 50K), and M2 macrophages (FIG.50L).

FIGS. 51A-51L depict the proportion amount of cells detected in mice ineach of the treatment groups in terminal lysed whole blood samples. FIG.51A depicts the number of TIL CD45+ cells as a percent of all live cellsdetected. FIGS. 51B-51L depict the number of total CD3+ cells (FIG.51B), CD4+ cells (FIG. 51C), CD8+ cells (FIG. 51D), CD3− non-T-cells(FIG. 51E), Tregs cells (FIG. 51F), proliferative T-cells (FIG. 51G),proliferative Tregs cells (FIG. 51H), CD11b+ cells (FIG. 51I), CD11c+cells (FIG. 51J), M1 macrophages (FIG. 51K), and M2 macrophages (FIG.51L).

FIG. 51M depicts the ratio of TIL M1/M2 macrophages in terminal lysedwhole blood samples in mice.

FIG. 52A-52E depict the proportion amount of LWB CD3+ cells detected inmice in each of the treatment groups in terminal lysed whole bloodsamples. FIG. 52A depicts the number of LWB CD3+ cells as a percent ofall CD45+ cells detected. FIGS. 52B-52E depict the number of totalCD3+TNFa+ cells (FIG. 52B), CD3+IFNg+ cells (FIG. 52C), CD3+IL6+ cells(FIG. 52D), and CD3+IL8+ cells (FIG. 52E) as a percent of all CD3+ cellsdetected.

FIG. 53A-53E depict the proportion amount of LWB CD4+ cells detected inmice in each of the treatment groups in terminal lysed whole bloodsamples. FIG. 53A depicts the number of LWB CD4+ cells as a percent ofall CD3+ cells detected. FIGS. 53B-53E depict the number of totalCD4+TNFa+ cells (FIG. 53B), CD4+IFNg+ cells (FIG. 53C), CD4+IL6+ cells(FIG. 53D), and CD4+IL8+ cells (FIG. 53E) as a percent of all CD4+ cellsdetected.

FIG. 54A-54E depict the proportion amount of LWB CD8+ cells detected inmice in each of the treatment groups in terminal lysed whole bloodsamples. FIG. 54A depicts the number of LWB CD8+ cells as a percent ofall CD3+ cells detected. FIGS. 54B-54E depict the number of totalCD8+TNFa+ cells (FIG. 54B), CD8+IFNg+ cells (FIG. 54C), CD8+IL6+ cells(FIG. 54D), and CD8+IL8+ cells (FIG. 54E) as a percent of all CD8+ cellsdetected.

FIG. 55A-55D depict the amount of MFI CD3+ cells detected in mice ineach of the treatment groups in terminal lysed whole blood samples.FIGS. 55A-55D depict the number of total CD3+TNFa+ cells (FIG. 55A),CD3+IFNg+ cells (FIG. 55B), CD3+IL6+ cells (FIG. 55C), and CD3+IL8+cells (FIG. 55D) as difference of all MFI cells detected.

FIG. 56A-56D depict the amount of MFI CD4+ cells detected in mice ineach of the treatment groups in terminal lysed whole blood samples.FIGS. 56A-56D depict the number of total CD4+TNFa+ cells (FIG. 56A),CD4+IFNg+ cells (FIG. 56B), CD4+IL6+ cells (FIG. 56C), and CD4+IL8+cells (FIG. 56D) as difference of all MFI cells detected.

FIG. 57A-57D depict the amount of MFI CD8+ cells detected in mice ineach of the treatment groups in terminal lysed whole blood samples.FIGS. 57A-57D depict the number of total CD8+TNFa+ cells (FIG. 57A),CD8+IFNg+ cells (FIG. 57B), CD8+IL6+ cells (FIG. 57C), and CD8+IL8+cells (FIG. 57D) as difference of all MFI cells detected.

FIG. 58A-58B depict the proportion amount of LWB CD8+ cells detected inmice in each of the treatment groups in interim lysed whole bloodsamples. FIGS. 58A-58B depict the number of CD8+ cells (FIG. 58A) andTregs cells (FIG. 58B) as a proportion of CD45+ cells.

FIG. 58C depicts the ratio of LWB CD8+/Treg cells in interim lysed wholeblood samples in mice.

FIG. 59A-59B depict the proportion amount of LWB CD8+ cells detected inmice in each of the treatment groups in terminal lysed whole bloodsamples. FIGS. 59A-59B depict the number of CD8+ cells (FIG. 59A) andTregs cells (FIG. 59B) as a proportion of CD45+ cells.

FIG. 59C depicts the ratio of LWB CD8+/Treg cells in terminal lysedwhole blood samples in mice.

FIG. 60A-60B depict the proportion amount of TIL CD8+ cells detected inmice in each of the treatment groups in terminal lysed whole bloodsamples. FIGS. 60A-60B depict the number of CD8+ cells (FIG. 60A) andTregs cells (FIG. 60B) as a proportion of CD45+ cells.

FIG. 60C depicts the ratio of TIL CD8+/Treg cells in terminal lysedwhole blood samples in mice.

DETAILED DESCRIPTION

The present disclosure employs, unless otherwise indicated, conventionalmolecular biology techniques, which are within the skill of the art.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art.

Definitions

Throughout this disclosure, various embodiments are presented in a rangeformat. It should be understood that the description in range format ismerely for convenience and brevity and should not be construed as aninflexible limitation on the scope of any embodiments. Accordingly, thedescription of a range should be considered to have specificallydisclosed all the possible subranges as well as individual numericalvalues within that range to the tenth of the unit of the lower limitunless the context clearly dictates otherwise. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual valueswithin that range, for example, 1.1, 2, 2.3, 5, and 5.9. This appliesregardless of the breadth of the range. The upper and lower limits ofthese intervening ranges may independently be included in the smallerranges, and are also encompassed within the disclosure, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the disclosure, unless thecontext clearly dictates otherwise.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of any embodiment.As used herein, the singular forms “a,” “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof. As used herein, the term “and/or”includes any and all combinations of one or more of the associatedlisted items.

Unless specifically stated or obvious from context, as used herein, theterm “about” in reference to a number or range of numbers is understoodto mean the stated number and numbers +/−10% thereof, or 10% below thelower listed limit and 10% above the higher listed limit for the valueslisted for a range.

Unless specifically stated, as used herein, the term “nucleic acid”encompasses double- or triple-stranded nucleic acids, as well assingle-stranded molecules. In double- or triple-stranded nucleic acids,the nucleic acid strands need not be coextensive (i.e., adouble-stranded nucleic acid need not be double-stranded along theentire length of both strands). Nucleic acid sequences, when provided,are listed in the 5′ to 3′ direction, unless stated otherwise. Methodsdescribed herein provide for the generation of isolated nucleic acids.Methods described herein additionally provide for the generation ofisolated and purified nucleic acids. A “nucleic acid” as referred toherein can comprise at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450,475, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600,1700, 1800, 1900, 2000, or more bases in length. Moreover, providedherein are methods for the synthesis of any number ofpolypeptide-segments encoding nucleotide sequences, including sequencesencoding non-ribosomal peptides (NRPs), sequences encoding non-ribosomalpeptide-synthetase (NRPS) modules and synthetic variants, polypeptidesegments of other modular proteins, such as antibodies, polypeptidesegments from other protein families, including non-coding DNA or RNA,such as regulatory sequences e.g. promoters, transcription factors,enhancers, siRNA, shRNA, RNAi, miRNA, small nucleolar RNA derived frommicroRNA, or any functional or structural DNA or RNA unit of interest.The following are non-limiting examples of polynucleotides: coding ornon-coding regions of a gene or gene fragment, intergenic DNA, loci(locus) defined from linkage analysis, exons, introns, messenger RNA(mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA),short-hairpin RNA (shRNA), micro-RNA (miRNA), small nucleolar RNA,ribozymes, complementary DNA (cDNA), which is a DNA representation ofmRNA, usually obtained by reverse transcription of messenger RNA (mRNA)or by amplification; DNA molecules produced synthetically or byamplification, genomic DNA, recombinant polynucleotides, branchedpolynucleotides, plasmids, vectors, isolated DNA of any sequence,isolated RNA of any sequence, nucleic acid probes, and primers. cDNAencoding for a gene or gene fragment referred herein may comprise atleast one region encoding for exon sequences without an interveningintron sequence in the genomic equivalent sequence.

Adenosine A2A and A2B Receptor Libraries

Provided herein are methods and compositions relating to Gprotein-coupled receptor (GPCR) binding libraries for adenosine A2Areceptor (ADORA2) comprising nucleic acids encoding for a scaffoldcomprising an adenosine A2A receptor binding domain. Scaffolds asdescribed herein can stably support an adenosine A2A receptor bindingdomain. The adenosine A2A receptor binding domain may be designed basedon surface interactions of an adenosine A2A receptor ligand andadenosine A2A receptor. Also provided herein are methods andcompositions relating to G protein-coupled receptor (GPCR) bindinglibraries for adenosine A2B receptor (ADORA2B) comprising nucleic acidsencoding for a scaffold comprising an adenosine A2B receptor bindingdomain. Scaffolds as described herein can stably support an adenosineA2B receptor binding domain. The adenosine A2B receptor binding domainmay be designed based on surface interactions of an adenosine A2Breceptor ligand and adenosine A2B receptor. Libraries as describedherein may be further variegated to provide for variant librariescomprising nucleic acids each encoding for a predetermined variant of atleast one predetermined reference nucleic acid sequence. Furtherdescribed herein are protein libraries that may be generated when thenucleic acid libraries are translated. In some instances, nucleic acidlibraries as described herein are transferred into cells to generate acell library. Also provided herein are downstream applications for thelibraries synthesized using methods described herein. Downstreamapplications include identification of variant nucleic acids or proteinsequences with enhanced biologically relevant functions, e.g., improvedstability, affinity, binding, functional activity, and for the treatmentor prevention of a disease state associated with adenosine A2A receptorsignaling, adenosine A2B receptor signaling, or both adenosine A2Areceptor signaling and adenosine A2B receptor signaling.

Methods, compositions, and systems described herein for the optimizationof adenosine A2A receptor immunoglobulins or antibodies, adenosine A2Breceptor immunoglobulins or antibodies, or both comprise a ratio-variantapproach that mirror the natural diversity of antibody sequences. Insome instances, libraries of optimized adenosine A2A receptorimmunoglobulins or antibodies comprise variant adenosine A2A receptorimmunoglobulin or antibody sequences. In some instances, the variantadenosine A2A receptor immunoglobulin or antibody sequences are designedcomprising variant CDR regions. In some instances, the variant adenosineA2A receptor immunoglobulin or antibody sequences comprising variant CDRregions are generated by shuffling the natural CDR sequences in a llama,humanized, or chimeric framework. In some instances, libraries ofoptimized adenosine A2B receptor immunoglobulins or antibodies comprisevariant adenosine A2B receptor immunoglobulin or antibody sequences. Insome instances, the variant adenosine A2B receptor immunoglobulin orantibody sequences are designed comprising variant CDR regions. In someinstances, the variant adenosine A2B receptor immunoglobulin or antibodysequences comprising variant CDR regions are generated by shuffling thenatural CDR sequences in a llama, humanized, or chimeric framework Insome instances, such libraries are synthesized, cloned into expressionvectors, and translation products (antibodies) evaluated for activity.In some instances, fragments of sequences are synthesized andsubsequently assembled. In some instances, expression vectors are usedto display and enrich desired antibodies, such as phage display. In someinstances, the phage vector is a Fab phagemid vector. Selectionpressures used during enrichment in some instances includes bindingaffinity, toxicity, immunological tolerance, stability, or other factor.Such expression vectors allow antibodies with specific properties to beselected (“panning”), and subsequent propagation or amplification ofsuch sequences enriches the library with these sequences. Panning roundscan be repeated any number of times, such as 1, 2, 3, 4, 5, 6, 7, ormore than 7 rounds. In some instances, each round of panning involves anumber of washes. In some instances, each round of panning involves atleast or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, ormore than 16 washes.

Described herein are methods and systems of in-silico library design.Libraries as described herein, in some instances, are designed based ona database comprising a variety of antibody sequences. In someinstances, the database comprises a plurality of variant antibodysequences against various targets. In some instances, the databasecomprises at least 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000,4500, 5000, or more than 5000 antibody sequences. An exemplary databaseis an iCAN database. In some instances, the database comprises naïve andmemory B-cell receptor sequences. In some instances, the naïve andmemory B-cell receptor sequences are human, mouse, or primate sequences.In some instances, the naïve and memory B-cell receptor sequences arehuman sequences. In some instances, the database is analyzed forposition specific variation. In some instances, antibodies describedherein comprise position specific variations in CDR regions. In someinstances, the CDR regions comprise multiple sites for variation.

Scaffold Libraries

Provided herein are libraries comprising nucleic acids encoding for ascaffold, wherein sequences for adenosine A2A receptor binding domainsare placed in the scaffold. Scaffolds described herein allow forimproved stability for a range of adenosine A2A receptor binding domainencoding sequences when inserted into the scaffold, as compared to anunmodified scaffold. Exemplary scaffolds include, but are not limitedto, a protein, a peptide, an immunoglobulin, derivatives thereof, orcombinations thereof. In some instances, the scaffold is animmunoglobulin. Scaffolds as described herein comprise improvedfunctional activity, structural stability, expression, specificity, or acombination thereof. In some instances, scaffolds comprise long regionsfor supporting an adenosine A2A receptor binding domain.

Provided herein are libraries comprising nucleic acids encoding for ascaffold, wherein sequences for adenosine A2B receptor binding domainsare placed in the scaffold. Scaffolds described herein allow forimproved stability for a range of adenosine A2B receptor binding domainencoding sequences when inserted into the scaffold, as compared to anunmodified scaffold. Exemplary scaffolds include, but are not limitedto, a protein, a peptide, an immunoglobulin, derivatives thereof, orcombinations thereof. In some instances, the scaffold is animmunoglobulin. Scaffolds as described herein comprise improvedfunctional activity, structural stability, expression, specificity, or acombination thereof. In some instances, scaffolds comprise long regionsfor supporting an adenosine A2B receptor binding domain.

Provided herein are libraries comprising nucleic acids encoding for ascaffold, wherein the scaffold is an immunoglobulin. In some instances,the immunoglobulin is an antibody. As used herein, the term antibodywill be understood to include proteins having the characteristictwo-armed, Y-shape of a typical antibody molecule as well as one or morefragments of an antibody that retain the ability to specifically bind toan antigen. Exemplary antibodies include, but are not limited to, amonoclonal antibody, a polyclonal antibody, a bi-specific antibody, amultispecific antibody, a grafted antibody, a human antibody, ahumanized antibody, a synthetic antibody, a chimeric antibody, acamelized antibody, a single-chain Fvs (scFv) (including fragments inwhich the VL and VH are joined using recombinant methods by a syntheticor natural linker that enables them to be made as a single protein chainin which the VL and VH regions pair to form monovalent molecules,including single chain Fab and scFab), a single chain antibody, a Fabfragment (including monovalent fragments comprising the VL, VH, CL, andCH1 domains), a F(ab′)2 fragment (including bivalent fragmentscomprising two Fab fragments linked by a disulfide bridge at the hingeregion), a Fd fragment (including fragments comprising the VH and CH1fragment), a Fv fragment (including fragments comprising the VL and VHdomains of a single arm of an antibody), a single-domain antibody (dAbor sdAb) (including fragments comprising a VH domain), an isolatedcomplementarity determining region (CDR), a diabody (including fragmentscomprising bivalent dimers such as two VL and VH domains bound to eachother and recognizing two different antigens), a fragment comprised ofonly a single monomeric variable domain, disulfide-linked Fvs (sdFv), anintrabody, an anti-idiotypic (anti-Id) antibody, or ab antigen-bindingfragments thereof. In some instances, the libraries disclosed hereincomprise nucleic acids encoding for a scaffold, wherein the scaffold isa Fv antibody, including Fv antibodies comprised of the minimum antibodyfragment which contains a complete antigen-recognition andantigen-binding site. In some embodiments, the Fv antibody consists of adimer of one heavy chain and one light chain variable domain in tight,non-covalent association, and the three hypervariable regions of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. In some embodiments, the six hypervariableregions confer antigen-binding specificity to the antibody. In someembodiments, a single variable domain (or half of an Fv comprising onlythree hypervariable regions specific for an antigen, including singledomain antibodies isolated from camelid animals comprising one heavychain variable domain or variable region of a heavy chain such as VHHantibodies or nanobodies) has the ability to recognize and bind antigen.In some instances, the libraries disclosed herein comprise nucleic acidsencoding for a scaffold, wherein the scaffold is a single-chain Fv orscFv, including antibody fragments comprising a VH, a VL, or both a VHand VL domain, wherein both domains are present in a single polypeptidechain. In some embodiments, the Fv polypeptide further comprises apolypeptide linker between the VH and VL domains allowing the scFv toform the desired structure for antigen binding. In some instances, ascFv is linked to the Fc fragment or a VHH is linked to the Fc fragment(including minibodies). In some instances, the antibody comprisesimmunoglobulin molecules and immunologically active fragments ofimmunoglobulin molecules, e.g., molecules that contain an antigenbinding site. Immunoglobulin molecules are of any type (e.g., IgG, IgE,IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG 2, IgG 3, IgG 4, IgA 1and IgA 2) or subclass.

In some embodiments, libraries comprise immunoglobulins that are adaptedto the species of an intended therapeutic target. Generally, thesemethods include “mammalization” and comprises methods for transferringdonor antigen-binding information to a less immunogenic mammal antibodyacceptor to generate useful therapeutic treatments. In some instances,the mammal is mouse, rat, equine, sheep, cow, primate (e.g., chimpanzee,baboon, gorilla, orangutan, monkey), dog, cat, pig, donkey, rabbit, andhuman. In some instances, provided herein are libraries and methods forfelinization and caninization of antibodies.

“Humanized” forms of non-human antibodies can be chimeric antibodiesthat contain minimal sequence derived from the non-human antibody. Ahumanized antibody is generally a human antibody (recipient antibody) inwhich residues from one or more CDRs are replaced by residues from oneor more CDRs of a non-human antibody (donor antibody). The donorantibody can be any suitable non-human antibody, such as a mouse, rat,rabbit, chicken, or non-human primate antibody having a desiredspecificity, affinity, or biological effect. In some instances, selectedframework region residues of the recipient antibody are replaced by thecorresponding framework region residues from the donor antibody.Humanized antibodies may also comprise residues that are not found ineither the recipient antibody or the donor antibody. In some instances,these modifications are made to further refine antibody performance.

“Caninization” can comprise a method for transferring non-canineantigen-binding information from a donor antibody to a less immunogeniccanine antibody acceptor to generate treatments useful as therapeuticsin dogs. In some instances, caninized forms of non-canine antibodiesprovided herein are chimeric antibodies that contain minimal sequencederived from non-canine antibodies. In some instances, caninizedantibodies are canine antibody sequences (“acceptor” or “recipient”antibody) in which hypervariable region residues of the recipient arereplaced by hypervariable region residues from a non-canine species(“donor” antibody) such as mouse, rat, rabbit, cat, dogs, goat, chicken,bovine, horse, llama, camel, dromedaries, sharks, non-human primates,human, humanized, recombinant sequence, or an engineered sequence havingthe desired properties. In some instances, framework region (FR)residues of the canine antibody are replaced by corresponding non-canineFR residues. In some instances, caninized antibodies include residuesthat are not found in the recipient antibody or in the donor antibody.In some instances, these modifications are made to further refineantibody performance. The caninized antibody may also comprise at leasta portion of an immunoglobulin constant region (Fc) of a canineantibody.

“Felinization” can comprise a method for transferring non-felineantigen-binding information from a donor antibody to a less immunogenicfeline antibody acceptor to generate treatments useful as therapeuticsin cats. In some instances, felinized forms of non-feline antibodiesprovided herein are chimeric antibodies that contain minimal sequencederived from non-feline antibodies. In some instances, felinizedantibodies are feline antibody sequences (“acceptor” or “recipient”antibody) in which hypervariable region residues of the recipient arereplaced by hypervariable region residues from a non-feline species(“donor” antibody) such as mouse, rat, rabbit, cat, dogs, goat, chicken,bovine, horse, llama, camel, dromedaries, sharks, non-human primates,human, humanized, recombinant sequence, or an engineered sequence havingthe desired properties. In some instances, framework region (FR)residues of the feline antibody are replaced by corresponding non-felineFR residues. In some instances, felinized antibodies include residuesthat are not found in the recipient antibody or in the donor antibody.In some instances, these modifications are made to further refineantibody performance. The felinized antibody may also comprise at leasta portion of an immunoglobulin constant region (Fc) of a felinizeantibody.

Provided herein are libraries comprising nucleic acids encoding for ascaffold, wherein the scaffold is a non-immunoglobulin. In someinstances, the scaffold is a non-immunoglobulin binding domain. Forexample, the scaffold is an antibody mimetic. Exemplary antibodymimetics include, but are not limited to, anticalins, affilins, affibodymolecules, affimers, affitins, alphabodies, avimers, atrimers, DARPins,fynomers, Kunitz domain-based proteins, monobodies, anticalins,knottins, armadillo repeat protein-based proteins, and bicyclicpeptides.

Libraries described herein comprising nucleic acids encoding for ascaffold, wherein the scaffold is an immunoglobulin, comprise variationsin at least one region of the immunoglobulin. Exemplary regions of theantibody for variation include, but are not limited to, acomplementarity-determining region (CDR), a variable domain, or aconstant domain. In some instances, the CDR is CDR1, CDR2, or CDR3. Insome instances, the CDR is a heavy domain including, but not limited to,CDRH1, CDRH2, and CDRH3. In some instances, the CDR is a light domainincluding, but not limited to, CDRL1, CDRL2, and CDRL3. In someinstances, the variable domain is variable domain, light chain (VL) orvariable domain, heavy chain (VH). In some instances, the VL domaincomprises kappa or lambda chains. In some instances, the constant domainis constant domain, light chain (CL) or constant domain, heavy chain(CH).

Methods described herein provide for synthesis of libraries comprisingnucleic acids encoding for a scaffold, wherein each nucleic acid encodesfor a predetermined variant of at least one predetermined referencenucleic acid sequence. In some cases, the predetermined referencesequence is a nucleic acid sequence encoding for a protein, and thevariant library comprises sequences encoding for variation of at least asingle codon such that a plurality of different variants of a singleresidue in the subsequent protein encoded by the synthesized nucleicacid are generated by standard translation processes. In some instances,the scaffold library comprises varied nucleic acids collectivelyencoding variations at multiple positions. In some instances, thevariant library comprises sequences encoding for variation of at least asingle codon of a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VL, or VHdomain. In some instances, the variant library comprises sequencesencoding for variation of multiple codons of a CDRH1, CDRH2, CDRH3,CDRL1, CDRL2, CDRL3, VL, or VH domain. In some instances, the variantlibrary comprises sequences encoding for variation of multiple codons offramework element 1 (FW1), framework element 2 (FW2), framework element3 (FW3), or framework element 4 (FW4). An exemplary number of codons forvariation include, but are not limited to, at least or about 1, 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,125, 150, 175, 225, 250, 275, 300, or more than 300 codons.

In some instances, the at least one region of the immunoglobulin forvariation is from heavy chain V-gene family, heavy chain D-gene family,heavy chain J-gene family, light chain V-gene family, or light chainJ-gene family. In some instances, the light chain V-gene familycomprises immunoglobulin kappa (IGK) gene or immunoglobulin lambda(IGL). Exemplary genes include, but are not limited to, IGHV1-18,IGHV1-69, IGHV1-8, IGHV3-21, IGHV3-23, IGHV3-30/33m, IGHV3-28, IGHV1-69,IGHV3-74, IGHV4-39, IGHV4-59/61, IGKV1-39, IGKV1-9, IGKV2-28, IGKV3-11,IGKV3-15, IGKV3-20, IGKV4-1, IGLV1-51, IGLV2-14, IGLV1-40, and IGLV3-1.In some instances, the gene is IGHV1-69, IGHV3-30, IGHV3-23, IGHV3,IGHV1-46, IGHV3-7, IGHV1, or IGHV1-8. In some instances, the gene isIGHV1-69 and IGHV3-30. In some instances, the gene is IGHJ3, IGHJ6,IGHJ, IGHJ4, IGHJ5, IGHJ2, or IGH1. In some instances, the gene isIGHJ3, IGHJ6, IGHJ, or IGHJ4.

Provided herein are libraries comprising nucleic acids encoding forimmunoglobulin scaffolds, wherein the libraries are synthesized withvarious numbers of fragments. In some instances, the fragments comprisethe CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VL, or VH domain. In someinstances, the fragments comprise framework element 1 (FW1), frameworkelement 2 (FW2), framework element 3 (FW3), or framework element 4(FW4). In some instances, the scaffold libraries are synthesized with atleast or about 2 fragments, 3 fragments, 4 fragments, 5 fragments, ormore than 5 fragments. The length of each of the nucleic acid fragmentsor average length of the nucleic acids synthesized may be at least orabout 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350,375, 400, 425, 450, 475, 500, 525, 550, 575, 600, or more than 600 basepairs. In some instances, the length is about 50 to 600, 75 to 575, 100to 550, 125 to 525, 150 to 500, 175 to 475, 200 to 450, 225 to 425, 250to 400, 275 to 375, or 300 to 350 base pairs.

Libraries comprising nucleic acids encoding for immunoglobulin scaffoldsas described herein comprise various lengths of amino acids whentranslated. In some instances, the length of each of the amino acidfragments or average length of the amino acid synthesized may be atleast or about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, ormore than 150 amino acids. In some instances, the length of the aminoacid is about 15 to 150, 20 to 145, 25 to 140, 30 to 135, 35 to 130, 40to 125, 45 to 120, 50 to 115, 55 to 110, 60 to 110, 65 to 105, 70 to100, or 75 to 95 amino acids. In some instances, the length of the aminoacid is about 22 amino acids to about 75 amino acids. In some instances,the immunoglobulin scaffolds comprise at least or about 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more than5000 amino acids.

A number of variant sequences for the at least one region of theimmunoglobulin for variation are de novo synthesized using methods asdescribed herein. In some instances, a number of variant sequences is denovo synthesized for CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VL, VH,or combinations thereof. In some instances, a number of variantsequences is de novo synthesized for framework element 1 (FW1),framework element 2 (FW2), framework element 3 (FW3), or frameworkelement 4 (FW4). The number of variant sequences may be at least orabout 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,425, 450, 475, 500, or more than 500 sequences. In some instances, thenumber of variant sequences is at least or about 500, 600, 700, 800,900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, or more than 8000sequences. In some instances, the number of variant sequences is about10 to 500, 25 to 475, 50 to 450, 75 to 425, 100 to 400, 125 to 375, 150to 350, 175 to 325, 200 to 300, 225 to 375, 250 to 350, or 275 to 325sequences.

Variant sequences for the at least one region of the immunoglobulin, insome instances, vary in length or sequence. In some instances, the atleast one region that is de novo synthesized is for CDRH1, CDRH2, CDRH3,CDRL1, CDRL2, CDRL3, VL, VH, or combinations thereof. In some instances,the at least one region that is de novo synthesized is for frameworkelement 1 (FW1), framework element 2 (FW2), framework element 3 (FW3),or framework element 4 (FW4). In some instances, the variant sequencecomprises at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,30, 35, 40, 45, 50, or more than 50 variant nucleotides or amino acidsas compared to wild-type. In some instances, the variant sequencecomprises at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,30, 35, 40, 45, or 50 additional nucleotides or amino acids as comparedto wild-type. In some instances, the variant sequence comprises at leastor about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or50 less nucleotides or amino acids as compared to wild-type. In someinstances, the libraries comprise at least or about 10¹, 10², 10³, 10⁴,10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, or more than 10¹⁰ variants.

Following synthesis of scaffold libraries, scaffold libraries may beused for screening and analysis. For example, scaffold libraries areassayed for library displayability and panning. In some instances,displayability is assayed using a selectable tag. Exemplary tagsinclude, but are not limited to, a radioactive label, a fluorescentlabel, an enzyme, a chemiluminescent tag, a colorimetric tag, anaffinity tag or other labels or tags that are known in the art. In someinstances, the tag is histidine, polyhistidine, myc, hemagglutinin (HA),or FLAG. In some instances, scaffold libraries are assayed by sequencingusing various methods including, but not limited to, single-moleculereal-time (SMRT) sequencing, Polony sequencing, sequencing by ligation,reversible terminator sequencing, proton detection sequencing, ionsemiconductor sequencing, nanopore sequencing, electronic sequencing,pyrosequencing, Maxam-Gilbert sequencing, chain termination (e.g.,Sanger) sequencing, +S sequencing, or sequencing by synthesis.

In some instances, the scaffold libraries are assayed for functionalactivity, structural stability (e.g., thermal stable or pH stable),expression, specificity, or a combination thereof. In some instances,the scaffold libraries are assayed for scaffolds capable of folding. Insome instances, a region of the antibody is assayed for functionalactivity, structural stability, expression, specificity, folding, or acombination thereof. For example, a VH region or VL region is assayedfor functional activity, structural stability, expression, specificity,folding, or a combination thereof.

Adenosine A2A Receptor Libraries

Provided herein are adenosine A2A receptor binding libraries comprisingnucleic acids encoding for scaffolds comprising sequences for adenosineA2A receptor binding domains. In some instances, the scaffolds areimmunoglobulins. In some instances, the scaffolds comprising sequencesfor adenosine A2A receptor binding domains are determined byinteractions between the adenosine A2A receptor binding domains and theadenosine A2A receptor.

Provided herein are libraries comprising nucleic acids encodingscaffolds comprising adenosine A2A receptor binding domains, wherein theadenosine A2A receptor binding domains are designed based on surfaceinteractions on adenosine A2A receptor. In some instances, the adenosineA2A receptor binding domain comprises a sequence as defined by SEQ IDNO: 1. In some instances, the adenosine A2A receptor binding domainsinteract with the amino- or carboxy-terminus of the adenosine A2Areceptor. In some instances, the adenosine A2A receptor binding domainsinteract with at least one transmembrane domain including, but notlimited to, transmembrane domain 1 (TM1), transmembrane domain 2 (TM2),transmembrane domain 3 (TM3), transmembrane domain 4 (TM4),transmembrane domain 5 (TM5), transmembrane domain 6 (TM6), andtransmembrane domain 7 (TM7). In some instances, the adenosine A2Areceptor binding domains interact with an intracellular surface of theadenosine A2A receptor. For example, the adenosine A2A receptor bindingdomains interact with at least one intracellular loop including, but notlimited to, intracellular loop 1 (ICL1), intracellular loop 2 (ICL2),and intracellular loop 3 (ICL3). In some instances, the adenosine A2Areceptor binding domains interact with an extracellular surface of theadenosine A2A receptor For example, the adenosine A2A receptor bindingdomains interact with at least one extracellular domain (ECD) orextracellular loop (ECL) of the adenosine A2A receptor. Theextracellular loops include, but are not limited to, extracellular loop1 (ECL1), extracellular loop 2 (ECL2), and extracellular loop 3 (ECL3).

Described herein are adenosine A2A receptor binding domains, wherein theadenosine A2A receptor binding domains are designed based on surfaceinteractions between an adenosine A2A receptor ligand and the adenosineA2A receptor. In some instances, the ligand is a peptide. In someinstances, the ligand is an adenosine A2A receptor agonist. In someinstances, the ligand is an adenosine A2A receptor antagonist. In someinstances, the ligand is an adenosine A2A receptor allosteric modulator.In some instances, the allosteric modulator is a negative allostericmodulator. In some instances, the allosteric modulator is a positiveallosteric modulator. Exemplary ligands of the adenosine A2A receptorinclude, but are not limited to DU172, PSB36, ZM241385, XAC, caffeine,T4G, T4E, 6DY, 6DZ, 6DX, 6DV, 8D1b, theophylline, UK-432097, adenosine,NECA, and CGS21680.

Sequences of adenosine A2A receptor binding domains based on surfaceinteractions between an adenosine A2A receptor ligand and the adenosineA2A receptor are analyzed using various methods. For example,multispecies computational analysis is performed. In some instances, astructure analysis is performed. In some instances, a sequence analysisis performed. Sequence analysis can be performed using a database knownin the art. Non-limiting examples of databases include, but are notlimited to, NCBI BLAST (blast.ncbi.nlm.nih.gov/Blast.cgi), UCSC GenomeBrowser (genome.ucsc.edu/), UniProt (www.uniprot.org/), and IUPHAR/BPSGuide to PHARMACOLOGY (guidetopharmacology.org/).

Described herein are adenosine A2A receptor binding domains designedbased on sequence analysis among various organisms. For example,sequence analysis is performed to identify homologous sequences indifferent organisms. Exemplary organisms include, but are not limitedto, mouse, rat, equine, sheep, cow, primate (e.g., chimpanzee, baboon,gorilla, orangutan, monkey), dog, cat, pig, donkey, rabbit, fish, fly,and human.

Following identification of adenosine A2A receptor binding domains,libraries comprising nucleic acids encoding for the adenosine A2Areceptor binding domains may be generated. In some instances, librariesof adenosine A2A receptor binding domains comprise sequences ofadenosine A2A receptor binding domains designed based on conformationalligand interactions, peptide ligand interactions, small molecule ligandinteractions, extracellular domains of adenosine A2A receptor, orantibodies that target adenosine A2A receptor. In some instances,libraries of adenosine A2A receptor binding domains comprise sequencesof adenosine A2A receptor binding domains designed based on peptideligand interactions. In some instances, the ligand is a not an antibodyligand. Libraries of adenosine A2A receptor binding domains may betranslated to generate protein libraries. In some instances, librariesof adenosine A2A receptor binding domains are translated to generatepeptide libraries, immunoglobulin libraries, derivatives thereof, orcombinations thereof. In some instances, libraries of adenosine A2Areceptor binding domains are translated to generate protein librariesthat are further modified to generate peptidomimetic libraries. In someinstances, libraries of adenosine A2A receptor binding domains aretranslated to generate protein libraries that are used to generate smallmolecules.

Methods described herein provide for synthesis of libraries of adenosineA2A receptor binding domains comprising nucleic acids each encoding fora predetermined variant of at least one predetermined reference nucleicacid sequence. In some cases, the predetermined reference sequence is anucleic acid sequence encoding for a protein, and the variant librarycomprises sequences encoding for variation of at least a single codonsuch that a plurality of different variants of a single residue in thesubsequent protein encoded by the synthesized nucleic acid are generatedby standard translation processes. In some instances, the libraries ofadenosine A2A receptor binding domains comprise varied nucleic acidscollectively encoding variations at multiple positions. In someinstances, the variant library comprises sequences encoding forvariation of at least a single codon in an adenosine A2A receptorbinding domain. In some instances, the variant library comprisessequences encoding for variation of multiple codons in an adenosine A2Areceptor binding domain. An exemplary number of codons for variationinclude, but are not limited to, at least or about 1, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150,175, 225, 250, 275, 300, or more than 300 codons.

Methods described herein provide for synthesis of libraries comprisingnucleic acids encoding for the adenosine A2A receptor binding domains,wherein the libraries comprise sequences encoding for variation oflength of the adenosine A2A receptor binding domains. In some instances,the library comprises sequences encoding for variation of length of atleast or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275, 300, or more than300 codons less as compared to a predetermined reference sequence. Insome instances, the library comprises sequences encoding for variationof length of at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250,275, 300, or more than 300 codons more as compared to a predeterminedreference sequence.

Following identification of adenosine A2A receptor binding domains, theadenosine A2A receptor binding domains may be placed in scaffolds asdescribed herein. In some instances, the scaffolds are immunoglobulins.In some instances, the adenosine A2A receptor binding domains are placedin the CDRH3 region. Adenosine A2A receptor binding domains that may beplaced in scaffolds can also be referred to as a motif. Scaffoldscomprising adenosine A2A receptor binding domains may be designed basedon binding, specificity, stability, expression, folding, or downstreamactivity. In some instances, the scaffolds comprising adenosine A2Areceptor binding domains enable contact with the adenosine A2A receptor.In some instances, the scaffolds comprising adenosine A2A receptorbinding domains enables high affinity binding with the adenosine A2Areceptor. An exemplary amino acid sequence of adenosine A2A receptorbinding domain is described in Table 1.

TABLE 1  Adenosine A2A receptor binding domain amino acid sequences SEQID NO GPCR Amino Acid Sequence 1 AdenosineMPIMGSSVYITVELAIAVLAILGNVLVCWAVWL A2A NSNLQNVTNYFVVSLAAADIAVGVLAIPFAITIreceptor STGFCAACHGCLFIACFVLVLTQSSIFSLLAIAIDRYIAIRIPLRYNGLVTGTRAKGIIAICWVLS FAIGLTPMLGWNNCGQPKEGKNHSQGCGEGQVACLFEDVVPMNYMVYFNFFACVLVPLLLMLGVYL RIFLAARRQLKQMESQPLPGERARSTLQKEVHAAKSLAIIVGLFALCWLPLHIINCFTFFCPDCSH APLWLMYLAIVLSHTNSVVNPFIYAYRIREFRQTFRKIIRSHVLRQQEPFKAAGTSARVLAAHGSD GEQVSLRLNGHPPGVWANGSAPHPERRPNGYALGLVSGGSAQESQGNTGLPDVELLSHELKGVCPE PPGLDDPLAQDGAGVS

Provided herein are scaffolds or immunoglobulins comprising adenosineA2A receptor binding domains, wherein the sequences of the adenosine A2Areceptor binding domains support interaction with adenosine A2Areceptor. The sequence may be homologous or identical to a sequence ofan adenosine A2A receptor ligand. In some instances, the adenosine A2Areceptor binding domain sequence comprises at least or about 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NO: 1. In some instances, the adenosine A2A receptorbinding domain sequence comprises at least or about 95% homology to SEQID NO: 1. In some instances, the adenosine A2A receptor binding domainsequence comprises at least or about 97% homology to SEQ ID NO: 1. Insome instances, the adenosine A2A receptor binding domain sequencecomprises at least or about 99% homology to SEQ ID NO: 1. In someinstances, the adenosine A2A receptor binding domain sequence comprisesat least or about 100% homology to SEQ ID NO: 1. In some instances, theadenosine A2A receptor binding domain sequence comprises at least aportion having at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370,380, 390, 400, or more than 400 amino acids of SEQ ID NO: 1.

Provided herein are antibodies or immunoglobulins, wherein the antibodyor immunoglobulin comprises a sequence at least or about 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to any one of SEQ ID NOs: 540-717. In some instances, theantibody or immunoglobulin sequence comprises at least or about 95%sequence identity to any one of SEQ ID NOs: 540-717. In some instances,the antibody or immunoglobulin sequence comprises at least or about 97%sequence identity to any one of SEQ ID NOs: 540-717. In some instances,the antibody or immunoglobulin sequence comprises at least or about 99%sequence identity to any one of SEQ ID NOs: 540-717. In some instances,the antibody or immunoglobulin sequence comprises at least or about 100%sequence identity to any one SEQ ID NOs: 540-717. In some instances, theantibody or immunoglobulin sequence comprises at least a portion havingat least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30,40, 50, 60, 70, 80, 90, 100, 110, or more than 110 amino acids of anyone of SEQ ID NOs: 540-717.

In some embodiments, the antibody or immunoglobulin sequence comprisescomplementarity determining regions (CDRs) comprising a sequence as setforth in Tables 15-16. In some embodiments, the antibody orimmunoglobulin sequence comprises complementarity determining regions(CDRs) comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one ofSEQ ID NOs: 6-539. In some instances, the antibody or immunoglobulinsequence comprises complementarity determining regions (CDRs) comprisingat least or about 95% homology to any one of SEQ ID NOs: 6-539. In someinstances, the antibody or immunoglobulin sequence comprisescomplementarity determining regions (CDRs) comprising at least or about97% homology to any one of SEQ ID NOs: 6-539. In some instances, theantibody or immunoglobulin sequence comprises complementaritydetermining regions (CDRs) comprising at least or about 99% homology toany one of SEQ ID NOs: 6-539. In some instances, the antibody orimmunoglobulin sequence comprises complementarity determining regions(CDRs) comprising at least or about 100% homology to any one of SEQ IDNOs: 6-539. In some instances, the antibody or immunoglobulin sequencecomprises complementarity determining regions (CDRs) comprising at leasta portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16,or more than 16 amino acids of any one of SEQ ID NOs: 6-539.

In some embodiments, the antibody or immunoglobulin sequence comprises aCDR1 comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one ofSEQ ID NOs: 6-94 or 273-361. In some instances, the antibody orimmunoglobulin sequence comprises CDR1 comprising at least or about 95%homology of any one of SEQ ID NOs: 6-94 and 273-361. In some instances,the antibody or immunoglobulin sequence comprises CDR1 comprising atleast or about 97% homology to any one of SEQ ID NOs: 6-94 or 273-361.In some instances, the antibody or immunoglobulin sequence comprisesCDR1 comprising at least or about 99% homology to any one of SEQ ID NOs:6-94 or 273-361. In some instances, the antibody or immunoglobulinsequence comprises CDR1 comprising at least or about 100% homology toany one of SEQ ID NOs: 6-270 or 273-537. In some instances, the antibodyor immunoglobulin sequence comprises CDR1 comprising at least a portionhaving at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or morethan 16 amino acids of any one of SEQ ID NOs: 6-94 or 273-361.

In some embodiments, the antibody or immunoglobulin sequence comprises aCDR2 comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one ofSEQ ID NOs: 95-183 and 362-450. In some instances, the antibody orimmunoglobulin sequence comprises CDR2 comprising at least or about 95%homology to any one of SEQ ID NOs: 95-183 and 362-450. In someinstances, the antibody or immunoglobulin sequence comprises CDR2comprising at least or about 97% homology to any one of SEQ ID NOs:795-183 and 362-450. In some instances, the antibody or immunoglobulinsequence comprises CDR2 comprising at least or about 99% homology to anyone of SEQ ID NOs: 95-183 and 362-450. In some instances, the antibodyor immunoglobulin sequence comprises CDR2 comprising at least or about100% homology to any one of SEQ ID NOs: 95-183 and 362-450. In someinstances, the antibody or immunoglobulin sequence comprises CDR2comprising at least a portion having at least or about 3, 4, 5, 6, 7, 8,9, 10, 12, 14, 16, or more than 16 amino acids of any one of SEQ ID NOs:95-183 and 362-450.

In some embodiments, the antibody or immunoglobulin sequence comprises aCDR3 comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one ofSEQ ID NOs: 184-272 and 451-539. In some instances, the antibody orimmunoglobulin sequence comprises CDR3 comprising at least or about 95%homology to any one of SEQ ID NOs: 184-272 and 451-539. In someinstances, the antibody or immunoglobulin sequence comprises CDR3comprising at least or about 97% homology to any one of SEQ ID NOs:184-272 and 451-539. In some instances, the antibody or immunoglobulinsequence comprises CDR3 comprising at least or about 99% homology to anyone of SEQ ID NOs: 184-272 and 451-539. In some instances, the antibodyor immunoglobulin sequence comprises CDR3 comprising at least or about100% homology to any one of SEQ ID NOs: 184-272 and 451-539. In someinstances, the antibody or immunoglobulin sequence comprises CDR3comprising at least a portion having at least or about 3, 4, 5, 6, 7, 8,9, 10, 12, 14, 16, or more than 16 amino acids of any one of SEQ ID NOs:184-272 and 451-539.

In some embodiments, the antibody or immunoglobulin sequence comprises aCDRH1 comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one ofSEQ ID NOs: 6-94; a CDRH2 comprising at least or about 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to any one of SEQ ID NOs: 95-183; and a CDRH3 comprising atleast or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 184-272.In some instances, the antibody or immunoglobulin sequence comprisesCDRH1 comprising at least or about 95%, 97%, 99%, or 100% homology toany one of SEQ ID NOs: 6-94; a CDRH2 comprising at least or about 95%,97%, 99%, or 100% homology to any one of SEQ ID NOs: 95-183; and a CDRH3comprising at least or about 95%, 97%, 99%, or 100% homology to any oneof SEQ ID NOs: 184-272. In some instances, the antibody orimmunoglobulin sequence comprises CDRH1 comprising at least a portionhaving at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or morethan 16 amino acids of SEQ ID NO: 6-94; a CDRH2 comprising at least aportion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, ormore than 16 amino acids of SEQ ID NO: 95-183; and a CDRH3 comprising atleast a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12,14, 16, or more than 16 amino acids of SEQ ID NO: 184-272.

In some embodiments, the antibody or immunoglobulin sequence comprises aCDRL1 comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:273-361; a CDRL2 comprising at least or about 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQID NO: 362-450; and a CDRL3 comprising at least or about 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NO: 451-539. In some instances, the antibody orimmunoglobulin sequence comprises CDRL1 comprising at least or about95%, 97%, 99%, or 100% homology to SEQ ID NO: 273-361; a CDRL2comprising at least or about 95%, 97%, 99%, or 100% homology to SEQ IDNO: 362-450; and a CDRL3 comprising at least or about 95%, 97%, 99%, or100% homology to SEQ ID NO: 451-539. In some instances, the antibody orimmunoglobulin sequence comprises CDRL1 comprising at least a portionhaving at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or morethan 16 amino acids of SEQ ID NO: 273-361; a CDRL2 comprising at least aportion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, ormore than 16 amino acids of SEQ ID NO: 362-450; and a CDRL3 comprisingat least a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12,14, 16, or more than 16 amino acids of SEQ ID NO: 451-539.

In some embodiments, the antibody or immunoglobulin sequence comprises aCDRH1 comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one ofSEQ ID NOs: 6-94; a CDRH2 comprising at least or about 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to any one of SEQ ID NOs: 95-183; a CDRH3 comprising at leastor about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to any one of SEQ ID NOs: 184-272, aCDRL1 comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one ofSEQ ID NOs: 273-362; a CDRL2 comprising at least or about 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to any one of SEQ ID NOs: 362-450; and a CDRL3 comprising atleast or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 451-539.In some instances, the antibody or immunoglobulin sequence comprisesCDRH1 comprising at least or about 95%, 97%, 99%, or 100% homology toany one of SEQ ID NOs: 6-94; a CDRH2 comprising at least or about 95%,97%, 99%, or 100% homology to any one of SEQ ID NOs: 95-183; a CDRH3comprising at least or about 95%, 97%, 99%, or 100% homology to any oneof SEQ ID NOs: 184-272; a CDRL1 comprising at least or about 95%, 97%,99%, or 100% homology to any one of SEQ ID NOs: 273-362; a CDRL2comprising at least or about 95%, 97%, 99%, or 100% homology to any oneof SEQ ID NOs: 362-450; and a CDRL3 comprising at least or about 95%,97%, 99%, or 100% homology to any one of SEQ ID NOs: 451-539. In someinstances, the antibody or immunoglobulin sequence comprises CDRH1comprising at least a portion having at least or about 3, 4, 5, 6, 7, 8,9, 10, 12, 14, 16, or more than 16 amino acids of any one of SEQ ID NOs:6-94; a CDRH2 comprising at least a portion having at least or about 3,4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16 amino acids of any oneof SEQ ID NOs: 95-183; a CDRH3 comprising at least a portion having atleast or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16amino acids of any one of SEQ ID NOs: 184-272; a CDRL1 comprising atleast a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12,14, 16, or more than 16 amino acids of any one of SEQ ID NOs: 273-362; aCDRL2 comprising at least a portion having at least or about 3, 4, 5, 6,7, 8, 9, 10, 12, 14, 16, or more than 16 amino acids of any one of SEQID NOs: 362-450; and a CDRL3 comprising at least a portion having atleast or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or more than 16amino acids of any one of SEQ ID NOs: 451-539.

Described herein, in some embodiments, are antibodies or immunoglobulinsthat bind to the adenosine A2A receptor. In some instances, theadenosine A2A receptor antibody or immunoglobulin sequence comprises aheavy chain variable domain comprising at least or about 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to any one of SEQ ID NOs: 540-628. In some instances, theadenosine A2A receptor antibody or immunoglobulin sequence comprises aheavy chain variable domain comprising at least or about 95% sequenceidentity to any one of SEQ ID NOs: 540-628. In some instances, theadenosine A2A receptor antibody or immunoglobulin sequence comprises aheavy chain variable domain comprising at least or about 97% sequenceidentity to any one of SEQ ID NOs: 540-628. In some instances, theadenosine A2A receptor antibody or immunoglobulin sequence comprises aheavy chain variable domain comprising at least or about 99% sequenceidentity to any one of SEQ ID NOs: 540-628. In some instances, theadenosine A2A receptor antibody or immunoglobulin sequence comprises aheavy chain variable domain comprising at least or about 100% sequenceidentity to any one of SEQ ID NOs: 540-628. In some instances, theadenosine A2A receptor antibody or immunoglobulin sequence comprises aheavy chain variable domain comprising at least a portion having atleast or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30,40, 50, 60, 70, 80, 90, 100, 110, or more than 110 amino acids of SEQ IDNOs: 540-628.

In some instances, the adenosine A2A receptor antibody or immunoglobulinsequence comprises a light chain variable domain comprising at least orabout 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% sequence identity to any one of SEQ ID NOs: 629-717. In someinstances, the adenosine A2A receptor antibody or immunoglobulinsequence comprises a light chain variable domain comprising at least orabout 95% sequence identity to any one of SEQ ID NOs: 629-717. In someinstances, the adenosine A2A receptor antibody or immunoglobulinsequence comprises a light chain variable domain comprising at least orabout 97% sequence identity to any one of SEQ ID NOs: 629-717. In someinstances, the adenosine A2A receptor antibody or immunoglobulinsequence comprises a light chain variable domain comprising at least orabout 99% sequence identity to any one of SEQ ID NOs: 629-717. In someinstances, the adenosine A2A receptor antibody or immunoglobulinsequence comprises a light chain variable domain comprising at least orabout 100% sequence identity to any one of SEQ ID NOs: 629-717. In someinstances, the adenosine A2A receptor antibody or immunoglobulinsequence comprises a light chain variable domain comprising at least aportion having at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14,16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290,300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, or more than 400amino acids of SEQ ID NOs: 629-717.

In some embodiments, the immunoglobulin heavy chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 540; and the immunoglobulin light chain comprises an amino acidsequence at least about 90% identical to that set forth in SEQ ID NO:629. In some embodiments, the immunoglobulin heavy chain comprises anamino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 541; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 630. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 542; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 631. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 543; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 632. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 544; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 633. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 545; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 634. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 546; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 635. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 547; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 636. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 548; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 637. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 549; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 638. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 550; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 639. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 551; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 640. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 552; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 641. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 553; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 642. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 554; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 643. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 555; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 644. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 556; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 645. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 557; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 646. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 558; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 647. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 559; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 648. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 560; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 649. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 561; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 650. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 562; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 651. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 563; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 652. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 564; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 653. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 565; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 654. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 566; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 655. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 567; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 656. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 568; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 657. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 569; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 658. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 570; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 659. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 571; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 660. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 572; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 661. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 573; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 662. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 574; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 663. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 575; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 664. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 576; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 665. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 577; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 666. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 578; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 667. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 579; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 668. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 580; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 669. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 581; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 670. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 582; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 671. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 583; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 672. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 584; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 673. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 585; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 674. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 586; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 675. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 587; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 676. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 588; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 677. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 589; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 678. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 590; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 679. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 591; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 680. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 592; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 681. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 593; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 682. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 594; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 683. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 595; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 684. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 596; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 685. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 597; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 686. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 598; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 687. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 599; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 688. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 600; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 689. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 601; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 690. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 602; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 691. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 603; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 692. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 604; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 693. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 605; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 694. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 606; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 695. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 607; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 696. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 608; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 697. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 609; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 698. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 610; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 699. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 611; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 700. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 612; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 701. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 613; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 702. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 614; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 703. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 615; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 704. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 616; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 705. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 617; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 706. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 618; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 707. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 619; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 708. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 620; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 709. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 621; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 710. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 622; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 711. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 623; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 712. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 624; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 713. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 625; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 714. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 626; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 715. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 627; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 716. In some embodiments, the immunoglobulin heavy chain comprisesan amino acid sequence at least about 90% identical to that set forth inSEQ ID NO: 628; and the immunoglobulin light chain comprises an aminoacid sequence at least about 90% identical to that set forth in SEQ IDNO: 717.

Provided herein are adenosine A2A receptor binding libraries comprisingnucleic acids encoding for scaffolds or immunoglobulins comprisingadenosine A2A receptor binding domains comprise variation in domaintype, domain length, or residue variation. In some instances, the domainis a region in the scaffold comprising the adenosine A2A receptorbinding domains. For example, the region is the VH, CDRH3, or VL domain.In some instances, the domain is the adenosine A2A receptor bindingdomain.

Methods described herein provide for synthesis of an adenosine A2Areceptor binding library of nucleic acids each encoding for apredetermined variant of at least one predetermined reference nucleicacid sequence. In some cases, the predetermined reference sequence is anucleic acid sequence encoding for a protein, and the variant librarycomprises sequences encoding for variation of at least a single codonsuch that a plurality of different variants of a single residue in thesubsequent protein encoded by the synthesized nucleic acid are generatedby standard translation processes. In some instances, the adenosine A2Areceptor binding library comprises varied nucleic acids collectivelyencoding variations at multiple positions. In some instances, thevariant library comprises sequences encoding for variation of at least asingle codon of a VH, CDRH3, or VL domain. In some instances, thevariant library comprises sequences encoding for variation of at least asingle codon in an adenosine A2A receptor binding domain. For example,at least one single codon of an adenosine A2A receptor binding domain aslisted in Table 1 is varied. In some instances, the variant librarycomprises sequences encoding for variation of multiple codons of a VH,CDRH3, or VL domain. In some instances, the variant library comprisessequences encoding for variation of multiple codons in an adenosine A2Areceptor binding domain. An exemplary number of codons for variationinclude, but are not limited to, at least or about 1, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150,175, 225, 250, 275, 300, or more than 300 codons.

Methods described herein provide for synthesis of an adenosine A2Areceptor binding library of nucleic acids each encoding for apredetermined variant of at least one predetermined reference nucleicacid sequence, wherein the adenosine A2A receptor binding librarycomprises sequences encoding for variation of length of a domain. Insome instances, the domain is VH, CDRH3, or VL domain. In someinstances, the domain is the adenosine A2A receptor binding domain. Insome instances, the library comprises sequences encoding for variationof length of at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275,300, or more than 300 codons less as compared to a predeterminedreference sequence. In some instances, the library comprises sequencesencoding for variation of length of at least or about 1, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125,150, 175, 200, 225, 250, 275, 300, or more than 300 codons more ascompared to a predetermined reference sequence.

Provided herein are adenosine A2A receptor binding libraries comprisingnucleic acids encoding for scaffolds comprising adenosine A2A receptorbinding domains, wherein the adenosine A2A receptor binding librariesare synthesized with various numbers of fragments. In some instances,the fragments comprise the VH, CDRH3, or VL domain. In some instances,the adenosine A2A receptor binding libraries are synthesized with atleast or about 2 fragments, 3 fragments, 4 fragments, 5 fragments, ormore than 5 fragments. The length of each of the nucleic acid fragmentsor average length of the nucleic acids synthesized may be at least orabout 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350,375, 400, 425, 450, 475, 500, 525, 550, 575, 600, or more than 600 basepairs. In some instances, the length is about 50 to 600, 75 to 575, 100to 550, 125 to 525, 150 to 500, 175 to 475, 200 to 450, 225 to 425, 250to 400, 275 to 375, or 300 to 350 base pairs.

Adenosine A2A receptor binding libraries comprising nucleic acidsencoding for scaffolds comprising adenosine A2A receptor binding domainsas described herein comprise various lengths of amino acids whentranslated. In some instances, the length of each of the amino acidfragments or average length of the amino acid synthesized may be atleast or about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, ormore than 150 amino acids. In some instances, the length of the aminoacid is about 15 to 150, 20 to 145, 25 to 140, 30 to 135, 35 to 130, 40to 125, 45 to 120, 50 to 115, 55 to 110, 60 to 110, 65 to 105, 70 to100, or 75 to 95 amino acids. In some instances, the length of the aminoacid is about 22 to about 75 amino acids.

Adenosine A2A receptor binding libraries comprising de novo synthesizedvariant sequences encoding for scaffolds comprising adenosine A2Areceptor binding domains comprise a number of variant sequences. In someinstances, a number of variant sequences is de novo synthesized for aCDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VL, VH, or a combinationthereof. In some instances, a number of variant sequences is de novosynthesized for framework element 1 (FW1), framework element 2 (FW2),framework element 3 (FW3), or framework element 4 (FW4). In someinstances, a number of variant sequences is de novo synthesized for anadenosine A2A receptor binding domain. For example, the number ofvariant sequences is about 1 to about 10 sequences for the VH domain,about 10⁸ sequences for the adenosine A2A receptor binding domain, andabout 1 to about 44 sequences for the VK domain. The number of variantsequences may be at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225,250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more than 500sequences. In some instances, the number of variant sequences is about10 to 300, 25 to 275, 50 to 250, 75 to 225, 100 to 200, or 125 to 150sequences.

Adenosine A2A receptor binding libraries comprising de novo synthesizedvariant sequences encoding for scaffolds comprising adenosine A2Areceptor binding domains comprise improved diversity. For example,variants are generated by placing adenosine A2A receptor binding domainvariants in immunoglobulin scaffold variants comprising N-terminal CDRH3variations and C-terminal CDRH3 variations. In some instances, variantsinclude affinity maturation variants. Alternatively or in combination,variants include variants in other regions of the immunoglobulinincluding, but not limited to, CDRH1, CDRH2, CDRL1, CDRL2, and CDRL3. Insome instances, the number of variants of the adenosine A2A receptorbinding libraries is least or about 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰,10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than10²⁰ non-identical sequences. For example, a library comprising about 10variant sequences for a VH region, about 237 variant sequences for aCDRH3 region, and about 43 variant sequences for a VL and CDRL3 regioncomprises 10⁵ non-identical sequences (10×237×43).

Provided herein are libraries comprising nucleic acids encoding for anadenosine A2A receptor antibody comprising variation in at least oneregion of the antibody, wherein the region is the CDR region. In someinstances, the adenosine A2A receptor antibody is a single domainantibody comprising one heavy chain variable domain such as a VHHantibody. In some instances, the VHH antibody comprises variation in oneor more CDR regions. In some instances, libraries described hereincomprise at least or about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800,2000, 2400, 2600, 2800, 3000, or more than 3000 sequences of a CDR1,CDR2, or CDR3. In some instances, libraries described herein comprise atleast or about 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³,10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than 10²⁰ sequences ofa CDR1, CDR2, or CDR3. For example, the libraries comprise at least 2000sequences of a CDR1, at least 1200 sequences for CDR2, and at least 1600sequences for CDR3. In some instances, each sequence is non-identical.

In some instances, the CDR1, CDR2, or CDR3 is of a variable domain,light chain (VL). CDR1, CDR2, or CDR3 of a variable domain, light chain(VL) can be referred to as CDRL1, CDRL2, or CDRL3, respectively. In someinstances, libraries described herein comprise at least or about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2400,2600, 2800, 3000, or more than 3000 sequences of a CDR1, CDR2, or CDR3of the VL. In some instances, libraries described herein comprise atleast or about 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³,10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than 10²⁰ sequences ofa CDR1, CDR2, or CDR3 of the VL. For example, the libraries comprise atleast 20 sequences of a CDR1 of the VL, at least 4 sequences of a CDR2of the VL, and at least 140 sequences of a CDR3 of the VL. In someinstances, the libraries comprise at least 2 sequences of a CDR1 of theVL, at least 1 sequence of CDR2 of the VL, and at least 3000 sequencesof a CDR3 of the VL. In some instances, the VL is IGKV1-39, IGKV1-9,IGKV2-28, IGKV3-11, IGKV3-15, IGKV3-20, IGKV4-1, IGLV1-51, IGLV2-14,IGLV1-40, or IGLV3-1. In some instances, the VL is IGKV2-28. In someinstances, the VL is IGLV1-51.

In some instances, the CDR1, CDR2, or CDR3 is of a variable domain,heavy chain (VH). CDR1, CDR2, or CDR3 of a variable domain, heavy chain(VH) can be referred to as CDRH1, CDRH2, or CDRH3, respectively. In someinstances, libraries described herein comprise at least or about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2400,2600, 2800, 3000, or more than 3000 sequences of a CDR1, CDR2, or CDR3of the VH. In some instances, libraries described herein comprise atleast or about 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³,10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than 10²⁰ sequences ofa CDR1, CDR2, or CDR3 of the VH. For example, the libraries comprise atleast 30 sequences of a CDR1 of the VH, at least 570 sequences of a CDR2of the VH, and at least 10⁸ sequences of a CDR3 of the VH. In someinstances, the libraries comprise at least 30 sequences of a CDR1 of theVH, at least 860 sequences of a CDR2 of the VH, and at least 10⁷sequences of a CDR3 of the VH. In some instances, the VH is IGHV1-18,IGHV1-69, IGHV1-8 IGHV3-21, IGHV3-23, IGHV3-30/33rn, IGHV3-28, IGHV3-74,IGHV4-39, or IGHV4-59/61. In some instances, the VH is IGHV1-69,IGHV3-30, IGHV3-23, IGHV3, IGHV1-46, IGHV3-7, IGHV1, or IGHV1-8. In someinstances, the VH is IGHV1-69 and IGHV3-30. In some instances, the VH isIGHV3-23.

Libraries as described herein, in some embodiments, comprise varyinglengths of a CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, or CDRH3. In someinstances, the length of the CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, or CDRH3comprises at least or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70,80, 90, or more than 90 amino acids in length. For example, the CDRH3comprises at least or about 12, 15, 16, 17, 20, 21, or 23 amino acids inlength. In some instances, the CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, orCDRH3 comprises a range of about 1 to about 10, about 5 to about 15,about 10 to about 20, or about 15 to about 30 amino acids in length.

Libraries comprising nucleic acids encoding for antibodies havingvariant CDR sequences as described herein comprise various lengths ofamino acids when translated. In some instances, the length of each ofthe amino acid fragments or average length of the amino acid synthesizedmay be at least or about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145,150, or more than 150 amino acids. In some instances, the length of theamino acid is about 15 to 150, 20 to 145, 25 to 140, 30 to 135, 35 to130, 40 to 125, 45 to 120, 50 to 115, 55 to 110, 60 to 110, 65 to 105,70 to 100, or 75 to 95 amino acids. In some instances, the length of theamino acid is about 22 amino acids to about 75 amino acids. In someinstances, the antibodies comprise at least or about 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more than 5000amino acids.

Ratios of the lengths of a CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, or CDRH3may vary in libraries described herein. In some instances, a CDRL1,CDRL2, CDRL3, CDRH1, CDRH2, or CDRH3 comprising at least or about 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or more than 90 amino acidsin length comprises about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,or more than 90% of the library. For example, a CDRH3 comprising about23 amino acids in length is present in the library at 40%, a CDRH3comprising about 21 amino acids in length is present in the library at30%, a CDRH3 comprising about 17 amino acids in length is present in thelibrary at 20%, and a CDRH3 comprising about 12 amino acids in length ispresent in the library at 10%. In some instances, a CDRH3 comprisingabout 20 amino acids in length is present in the library at 40%, a CDRH3comprising about 16 amino acids in length is present in the library at30%, a CDRH3 comprising about 15 amino acids in length is present in thelibrary at 20%, and a CDRH3 comprising about 12 amino acids in length ispresent in the library at 10%.

Libraries as described herein encoding for a VHH antibody comprisevariant CDR sequences that are shuffled to generate a library with atheoretical diversity of at least or about 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹,10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than 10²⁰sequences. In some instances, the library has a final library diversityof at least or about 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵,10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than 10²⁰ sequences.

Provided herein are adenosine A2A receptor binding libraries encodingfor an immunoglobulin. In some instances, the adenosine A2A receptorimmunoglobulin is an antibody. In some instances, the adenosine A2Areceptor immunoglobulin is a VHH antibody. In some instances, theadenosine A2A receptor immunoglobulin comprises a binding affinity(e.g., K_(D)) to adenosine A2A receptor of less than 1 nM, less than 1.2nM, less than 2 nM, less than 5 nM, less than 10 nM, less than 11 nm,less than 13.5 nM, less than 15 nM, less than 20 nM, less than 25 nM, orless than 30 nM. In some instances, the adenosine A2A receptorimmunoglobulin comprises a K_(D) of less than 1 nM. In some instances,the adenosine A2A receptor immunoglobulin comprises a K_(D) of less than1.2 nM. In some instances, the adenosine A2A receptor immunoglobulincomprises a K_(D) of less than 2 nM. In some instances, the adenosineA2A receptor immunoglobulin comprises a K_(D) of less than 5 nM. In someinstances, the adenosine A2A receptor immunoglobulin comprises a K_(D)of less than 10 nM. In some instances, the adenosine A2A receptorimmunoglobulin comprises a K_(D) of less than 13.5 nM. In someinstances, the adenosine A2A receptor immunoglobulin comprises a K_(D)of less than 15 nM. In some instances, the adenosine A2A receptorimmunoglobulin comprises a K_(D) of less than 20 nM. In some instances,the adenosine A2A receptor immunoglobulin comprises a K_(D) of less than25 nM. In some instances, the adenosine A2A receptor immunoglobulincomprises a K_(D) of less than 30 nM.

In some instances, the adenosine A2A receptor immunoglobulin is anadenosine A2A receptor agonist. In some instances, the adenosine A2Areceptor immunoglobulin is an adenosine A2A receptor antagonist. In someinstances, the adenosine A2A receptor immunoglobulin is an adenosine A2Areceptor allosteric modulator. In some instances, the allostericmodulator is a negative allosteric modulator. In some instances, theallosteric modulator is a positive allosteric modulator. In someinstances, the adenosine A2A receptor immunoglobulin results inagonistic, antagonistic, or allosteric effects at a concentration of atleast or about 1 nM, 2 nM, 4 nM, 6 nM, 8 nM, 10 nM, 20 nM, 30 nM, 40 nM,50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 120 nM, 140 nM, 160 nM, 180nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1000nM, or more than 1000 nM. In some instances, the adenosine A2A receptorimmunoglobulin is a negative allosteric modulator. In some instances,the adenosine A2A receptor immunoglobulin is a negative allostericmodulator at a concentration of at least or about 0.001, 0.005, 0.01,0.05, 0.1, 0.5, 1 nM, 2 nM, 4 nM, 6 nM, 8 nM, 10 nM, 20 nM, 30 nM, 40nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, or more than 100 nM. Insome instances, the adenosine A2A receptor immunoglobulin is a negativeallosteric modulator at a concentration in a range of about 0.001 toabout 100, 0.01 to about 90, about 0.1 to about 80, 1 to about 50, about10 to about 40 nM, or about 1 to about 10 nM. In some instances, theadenosine A2A receptor immunoglobulin comprises an EC50 or IC50 of atleast or about 0.001, 0.0025, 0.005, 0.01, 0.025, 0.05, 0.06, 0.07,0.08, 0.9, 0.1, 0.5, 1, 2, 3, 4, 5, 6, or more than 6 nM. In someinstances, the adenosine A2A receptor immunoglobulin comprises an EC50or IC50 of at least or about 1 nM, 2 nM, 4 nM, 6 nM, 8 nM, 10 nM, 20 nM,30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, or more than100 nM.

Adenosine A2A receptor immunoglobulins as described herein may compriseimproved properties. In some instances, the adenosine A2A receptorimmunoglobulins are monomeric. In some instances, the adenosine A2Areceptor immunoglobulins are not prone to aggregation. In someinstances, at least or about 70%, 75%, 80%, 85%, 90%, 95%, or 99% of theadenosine A2A receptor immunoglobulins are monomeric. In some instances,the adenosine A2A receptor immunoglobulins are thermostable. In someinstances, the adenosine A2A receptor immunoglobulins result in reducednon-specific binding.

Following synthesis of adenosine A2A receptor binding librariescomprising nucleic acids encoding scaffolds comprising adenosine A2Areceptor binding domains, libraries may be used for screening andanalysis. For example, libraries are assayed for library displayabilityand panning. In some instances, displayability is assayed using aselectable tag. Exemplary tags include, but are not limited to, aradioactive label, a fluorescent label, an enzyme, a chemiluminescenttag, a colorimetric tag, an affinity tag or other labels or tags thatare known in the art. In some instances, the tag is histidine,polyhistidine, myc, hemagglutinin (HA), or FLAG. The adenosine A2Areceptor binding libraries may comprise nucleic acids encoding scaffoldscomprising adenosine A2A receptor binding domains with multiple tagssuch as GFP, FLAG, and Lucy as well as a DNA barcode. In some instances,libraries are assayed by sequencing using various methods including, butnot limited to, single-molecule real-time (SMRT) sequencing, Polonysequencing, sequencing by ligation, reversible terminator sequencing,proton detection sequencing, ion semiconductor sequencing, nanoporesequencing, electronic sequencing, pyrosequencing, Maxam-Gilbertsequencing, chain termination (e.g., Sanger) sequencing, +S sequencing,or sequencing by synthesis.

Adenosine A2B Receptor Libraries

Provided herein are adenosine A2B receptor binding libraries comprisingnucleic acids encoding for scaffolds comprising sequences for adenosineA2B receptor binding domains. In some instances, the scaffolds areimmunoglobulins. In some instances, the scaffolds comprising sequencesfor adenosine A2B receptor binding domains are determined byinteractions between the adenosine A2B receptor binding domains and theadenosine A2B receptor.

Provided herein are libraries comprising nucleic acids encodingscaffolds comprising adenosine A2B receptor binding domains, wherein theadenosine A2B receptor binding domains are designed based on surfaceinteractions on adenosine A2B receptor. In some instances, the adenosineA2B receptor binding domains interact with the amino- orcarboxy-terminus of the adenosine A2B receptor. In some instances, theadenosine A2B receptor binding domains interact with at least onetransmembrane domain including, but not limited to, transmembrane domain1 (TM1), transmembrane domain 2 (TM2), transmembrane domain 3 (TM3),transmembrane domain 4 (TM4), transmembrane domain 5 (TM5),transmembrane domain 6 (TM6), and transmembrane domain 7 (TM7). In someinstances, the adenosine A2B receptor binding domains interact with anintracellular surface of the adenosine A2B receptor. For example, theadenosine A2B receptor binding domains interact with at least oneintracellular loop including, but not limited to, intracellular loop 1(ICL1), intracellular loop 2 (ICL2), and intracellular loop 3 (ICL3). Insome instances, the adenosine A2B receptor binding domains interact withan extracellular surface of the adenosine A2B receptor For example, theadenosine A2B receptor binding domains interact with at least oneextracellular domain (ECD) or extracellular loop (ECL) of the adenosineA2B receptor. The extracellular loops include, but are not limited to,extracellular loop 1 (ECL1), extracellular loop 2 (ECL2), andextracellular loop 3 (ECL3).

Described herein are adenosine A2B receptor binding domains, wherein theadenosine A2B receptor binding domains are designed based on surfaceinteractions between an adenosine A2B receptor ligand and the adenosineA2B receptor. In some instances, the ligand is a peptide. In someinstances, the ligand is an adenosine A2B receptor agonist. In someinstances, the ligand is an adenosine A2B receptor antagonist. In someinstances, the ligand is an adenosine A2B receptor allosteric modulator.In some instances, the allosteric modulator is a negative allostericmodulator. In some instances, the allosteric modulator is a positiveallosteric modulator. Exemplary ligands of the adenosine A2B receptorinclude, but are not limited to DU172, PSB36, ZM241385, XAC, caffeine,T4G, T4E, 6DY, 6DZ, 6DX, 6DV, 8D1b, theophylline, UK-432097, adenosine,NECA, and CGS21680.

Sequences of adenosine A2B receptor binding domains based on surfaceinteractions between an adenosine A2B receptor ligand and the adenosineA2B receptor are analyzed using various methods. For example,multispecies computational analysis is performed. In some instances, astructure analysis is performed. In some instances, a sequence analysisis performed. Sequence analysis can be performed using a database knownin the art. Non-limiting examples of databases include, but are notlimited to, NCBI BLAST (blast.ncbi.nlm.nih.gov/Blast.cgi), UCSC GenomeBrowser (genome.ucsc.edu/), UniProt (www.uniprot.org/), and IUPHAR/BPSGuide to PHARMACOLOGY (guidetopharmacology.org/).

Described herein are adenosine A2B receptor binding domains designedbased on sequence analysis among various organisms. For example,sequence analysis is performed to identify homologous sequences indifferent organisms. Exemplary organisms include, but are not limitedto, mouse, rat, equine, sheep, cow, primate (e.g., chimpanzee, baboon,gorilla, orangutan, monkey), dog, cat, pig, donkey, rabbit, fish, fly,and human.

Following identification of adenosine A2B receptor binding domains,libraries comprising nucleic acids encoding for the adenosine A2Breceptor binding domains may be generated. In some instances, librariesof adenosine A2B receptor binding domains comprise sequences ofadenosine A2B receptor binding domains designed based on conformationalligand interactions, peptide ligand interactions, small molecule ligandinteractions, extracellular domains of adenosine A2B receptor, orantibodies that target adenosine A2B receptor. In some instances,libraries of adenosine A2B receptor binding domains comprise sequencesof adenosine A2B receptor binding domains designed based on peptideligand interactions. In some instances, the ligand is a not an antibodyligand. Libraries of adenosine A2B receptor binding domains may betranslated to generate protein libraries. In some instances, librariesof adenosine A2B receptor binding domains are translated to generatepeptide libraries, immunoglobulin libraries, derivatives thereof, orcombinations thereof. In some instances, libraries of adenosine A2Breceptor binding domains are translated to generate protein librariesthat are further modified to generate peptidomimetic libraries. In someinstances, libraries of adenosine A2B receptor binding domains aretranslated to generate protein libraries that are used to generate smallmolecules.

Methods described herein provide for synthesis of libraries of adenosineA2B receptor binding domains comprising nucleic acids each encoding fora predetermined variant of at least one predetermined reference nucleicacid sequence. In some cases, the predetermined reference sequence is anucleic acid sequence encoding for a protein, and the variant librarycomprises sequences encoding for variation of at least a single codonsuch that a plurality of different variants of a single residue in thesubsequent protein encoded by the synthesized nucleic acid are generatedby standard translation processes. In some instances, the libraries ofadenosine A2B receptor binding domains comprise varied nucleic acidscollectively encoding variations at multiple positions. In someinstances, the variant library comprises sequences encoding forvariation of at least a single codon in an adenosine A2B receptorbinding domain. In some instances, the variant library comprisessequences encoding for variation of multiple codons in an adenosine A2Breceptor binding domain. An exemplary number of codons for variationinclude, but are not limited to, at least or about 1, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150,175, 225, 250, 275, 300, or more than 300 codons.

Methods described herein provide for synthesis of libraries comprisingnucleic acids encoding for the adenosine A2B receptor binding domains,wherein the libraries comprise sequences encoding for variation oflength of the adenosine A2B receptor binding domains. In some instances,the library comprises sequences encoding for variation of length of atleast or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275, 300, or more than300 codons less as compared to a predetermined reference sequence. Insome instances, the library comprises sequences encoding for variationof length of at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250,275, 300, or more than 300 codons more as compared to a predeterminedreference sequence.

Following identification of adenosine A2B receptor binding domains, theadenosine A2B receptor binding domains may be placed in scaffolds asdescribed herein. In some instances, the scaffolds are immunoglobulins.In some instances, the adenosine A2B receptor binding domains are placedin the CDRH3 region. Adenosine A2B receptor binding domains that may beplaced in scaffolds can also be referred to as a motif. Scaffoldscomprising adenosine A2B receptor binding domains may be designed basedon binding, specificity, stability, expression, folding, or downstreamactivity. In some instances, the scaffolds comprising adenosine A2Breceptor binding domains enable contact with the adenosine A2B receptor.In some instances, the scaffolds comprising adenosine A2B receptorbinding domains enables high affinity binding with the adenosine A2Breceptor.

Described herein, in some embodiments, are antibodies or immunoglobulinsthat bind to the adenosine A2B receptor.

Provided herein are adenosine A2B receptor binding libraries comprisingnucleic acids encoding for scaffolds or immunoglobulins comprisingadenosine A2B receptor binding domains comprise variation in domaintype, domain length, or residue variation. In some instances, the domainis a region in the scaffold comprising the adenosine A2B receptorbinding domains. For example, the region is the VH, CDRH3, or VL domain.In some instances, the domain is the adenosine A2B receptor bindingdomain.

Methods described herein provide for synthesis of an adenosine A2Breceptor binding library of nucleic acids each encoding for apredetermined variant of at least one predetermined reference nucleicacid sequence. In some cases, the predetermined reference sequence is anucleic acid sequence encoding for a protein, and the variant librarycomprises sequences encoding for variation of at least a single codonsuch that a plurality of different variants of a single residue in thesubsequent protein encoded by the synthesized nucleic acid are generatedby standard translation processes. In some instances, the adenosine A2Breceptor binding library comprises varied nucleic acids collectivelyencoding variations at multiple positions. In some instances, thevariant library comprises sequences encoding for variation of at least asingle codon of a VH, CDRH3, or VL domain. In some instances, thevariant library comprises sequences encoding for variation of at least asingle codon in an adenosine A2B receptor binding domain. In someinstances, the variant library comprises sequences encoding forvariation of multiple codons of a VH, CDRH3, or VL domain. In someinstances, the variant library comprises sequences encoding forvariation of multiple codons in an adenosine A2B receptor bindingdomain. An exemplary number of codons for variation include, but are notlimited to, at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275,300, or more than 300 codons.

Methods described herein provide for synthesis of an adenosine A2Breceptor binding library of nucleic acids each encoding for apredetermined variant of at least one predetermined reference nucleicacid sequence, wherein the adenosine A2B receptor binding librarycomprises sequences encoding for variation of length of a domain. Insome instances, the domain is VH, CDRH3, or VL domain. In someinstances, the domain is the adenosine A2B receptor binding domain. Insome instances, the library comprises sequences encoding for variationof length of at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275,300, or more than 300 codons less as compared to a predeterminedreference sequence. In some instances, the library comprises sequencesencoding for variation of length of at least or about 1, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125,150, 175, 200, 225, 250, 275, 300, or more than 300 codons more ascompared to a predetermined reference sequence.

Provided herein are adenosine A2B receptor binding libraries comprisingnucleic acids encoding for scaffolds comprising adenosine A2B receptorbinding domains, wherein the adenosine A2B receptor binding librariesare synthesized with various numbers of fragments. In some instances,the fragments comprise the VH, CDRH3, or VL domain. In some instances,the adenosine A2B receptor binding libraries are synthesized with atleast or about 2 fragments, 3 fragments, 4 fragments, 5 fragments, ormore than 5 fragments. The length of each of the nucleic acid fragmentsor average length of the nucleic acids synthesized may be at least orabout 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350,375, 400, 425, 450, 475, 500, 525, 550, 575, 600, or more than 600 basepairs. In some instances, the length is about 50 to 600, 75 to 575, 100to 550, 125 to 525, 150 to 500, 175 to 475, 200 to 450, 225 to 425, 250to 400, 275 to 375, or 300 to 350 base pairs.

Adenosine A2B receptor binding libraries comprising nucleic acidsencoding for scaffolds comprising adenosine A2B receptor binding domainsas described herein comprise various lengths of amino acids whentranslated. In some instances, the length of each of the amino acidfragments or average length of the amino acid synthesized may be atleast or about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, ormore than 150 amino acids. In some instances, the length of the aminoacid is about 15 to 150, 20 to 145, 25 to 140, 30 to 135, 35 to 130, 40to 125, 45 to 120, 50 to 115, 55 to 110, 60 to 110, 65 to 105, 70 to100, or 75 to 95 amino acids. In some instances, the length of the aminoacid is about 22 to about 75 amino acids.

Adenosine A2B receptor binding libraries comprising de novo synthesizedvariant sequences encoding for scaffolds comprising adenosine A2Breceptor binding domains comprise a number of variant sequences. In someinstances, a number of variant sequences is de novo synthesized for aCDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, VL, VH, or a combinationthereof. In some instances, a number of variant sequences is de novosynthesized for framework element 1 (FW1), framework element 2 (FW2),framework element 3 (FW3), or framework element 4 (FW4). In someinstances, a number of variant sequences is de novo synthesized for anadenosine A2B receptor binding domain. For example, the number ofvariant sequences is about 1 to about 10 sequences for the VH domain,about 10⁸ sequences for the adenosine A2B receptor binding domain, andabout 1 to about 44 sequences for the VK domain. The number of variantsequences may be at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225,250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more than 500sequences. In some instances, the number of variant sequences is about10 to 300, 25 to 275, 50 to 250, 75 to 225, 100 to 200, or 125 to 150sequences.

Adenosine A2B receptor binding libraries comprising de novo synthesizedvariant sequences encoding for scaffolds comprising adenosine A2Breceptor binding domains comprise improved diversity. For example,variants are generated by placing adenosine A2B receptor binding domainvariants in immunoglobulin scaffold variants comprising N-terminal CDRH3variations and C-terminal CDRH3 variations. In some instances, variantsinclude affinity maturation variants. Alternatively or in combination,variants include variants in other regions of the immunoglobulinincluding, but not limited to, CDRH1, CDRH2, CDRL1, CDRL2, and CDRL3. Insome instances, the number of variants of the adenosine A2B receptorbinding libraries is least or about 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰,10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than10²⁰ non-identical sequences. For example, a library comprising about 10variant sequences for a VH region, about 237 variant sequences for aCDRH3 region, and about 43 variant sequences for a VL and CDRL3 regioncomprises 10⁵ non-identical sequences (10×237×43).

Provided herein are libraries comprising nucleic acids encoding for anadenosine A2B receptor antibody comprising variation in at least oneregion of the antibody, wherein the region is the CDR region. In someinstances, the adenosine A2B receptor antibody is a single domainantibody comprising one heavy chain variable domain such as a VHHantibody. In some instances, the VHH antibody comprises variation in oneor more CDR regions. In some instances, libraries described hereincomprise at least or about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800,2000, 2400, 2600, 2800, 3000, or more than 3000 sequences of a CDR1,CDR2, or CDR3. In some instances, libraries described herein comprise atleast or about 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³,10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than 10²⁰ sequences ofa CDR1, CDR2, or CDR3. For example, the libraries comprise at least 2000sequences of a CDR1, at least 1200 sequences for CDR2, and at least 1600sequences for CDR3. In some instances, each sequence is non-identical.

In some instances, the CDR1, CDR2, or CDR3 is of a variable domain,light chain (VL). CDR1, CDR2, or CDR3 of a variable domain, light chain(VL) can be referred to as CDRL1, CDRL2, or CDRL3, respectively. In someinstances, libraries described herein comprise at least or about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2400,2600, 2800, 3000, or more than 3000 sequences of a CDR1, CDR2, or CDR3of the VL. In some instances, libraries described herein comprise atleast or about 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³,10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than 10²⁰ sequences ofa CDR1, CDR2, or CDR3 of the VL. For example, the libraries comprise atleast 20 sequences of a CDR1 of the VL, at least 4 sequences of a CDR2of the VL, and at least 140 sequences of a CDR3 of the VL. In someinstances, the libraries comprise at least 2 sequences of a CDR1 of theVL, at least 1 sequence of CDR2 of the VL, and at least 3000 sequencesof a CDR3 of the VL. In some instances, the VL is IGKV1-39, IGKV1-9,IGKV2-28, IGKV3-11, IGKV3-15, IGKV3-20, IGKV4-1, IGLV1-51, IGLV2-14,IGLV1-40, or IGLV3-1. In some instances, the VL is IGKV2-28. In someinstances, the VL is IGLV1-51.

In some instances, the CDR1, CDR2, or CDR3 is of a variable domain,heavy chain (VH). CDR1, CDR2, or CDR3 of a variable domain, heavy chain(VH) can be referred to as CDRH1, CDRH2, or CDRH3, respectively. In someinstances, libraries described herein comprise at least or about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2400,2600, 2800, 3000, or more than 3000 sequences of a CDR1, CDR2, or CDR3of the VH. In some instances, libraries described herein comprise atleast or about 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³,10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than 10²⁰ sequences ofa CDR1, CDR2, or CDR3 of the VH. For example, the libraries comprise atleast 30 sequences of a CDR1 of the VH, at least 570 sequences of a CDR2of the VH, and at least 10⁸ sequences of a CDR3 of the VH. In someinstances, the libraries comprise at least 30 sequences of a CDR1 of theVH, at least 860 sequences of a CDR2 of the VH, and at least 10⁷sequences of a CDR3 of the VH. In some instances, the VH is IGHV1-18,IGHV1-69, IGHV1-8 IGHV3-21, IGHV3-23, IGHV3-30/33rn, IGHV3-28, IGHV3-74,IGHV4-39, or IGHV4-59/61. In some instances, the VH is IGHV1-69,IGHV3-30, IGHV3-23, IGHV3, IGHV1-46, IGHV3-7, IGHV1, or IGHV1-8. In someinstances, the VH is IGHV1-69 and IGHV3-30. In some instances, the VH isIGHV3-23.

Libraries as described herein, in some embodiments, comprise varyinglengths of a CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, or CDRH3. In someinstances, the length of the CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, or CDRH3comprises at least or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70,80, 90, or more than 90 amino acids in length. For example, the CDRH3comprises at least or about 12, 15, 16, 17, 20, 21, or 23 amino acids inlength. In some instances, the CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, orCDRH3 comprises a range of about 1 to about 10, about 5 to about 15,about 10 to about 20, or about 15 to about 30 amino acids in length.

Libraries comprising nucleic acids encoding for antibodies havingvariant CDR sequences as described herein comprise various lengths ofamino acids when translated. In some instances, the length of each ofthe amino acid fragments or average length of the amino acid synthesizedmay be at least or about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145,150, or more than 150 amino acids. In some instances, the length of theamino acid is about 15 to 150, 20 to 145, 25 to 140, 30 to 135, 35 to130, 40 to 125, 45 to 120, 50 to 115, 55 to 110, 60 to 110, 65 to 105,70 to 100, or 75 to 95 amino acids. In some instances, the length of theamino acid is about 22 amino acids to about 75 amino acids. In someinstances, the antibodies comprise at least or about 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more than 5000amino acids.

Ratios of the lengths of a CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, or CDRH3may vary in libraries described herein. In some instances, a CDRL1,CDRL2, CDRL3, CDRH1, CDRH2, or CDRH3 comprising at least or about 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or more than 90 amino acidsin length comprises about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,or more than 90% of the library. For example, a CDRH3 comprising about23 amino acids in length is present in the library at 40%, a CDRH3comprising about 21 amino acids in length is present in the library at30%, a CDRH3 comprising about 17 amino acids in length is present in thelibrary at 20%, and a CDRH3 comprising about 12 amino acids in length ispresent in the library at 10%. In some instances, a CDRH3 comprisingabout 20 amino acids in length is present in the library at 40%, a CDRH3comprising about 16 amino acids in length is present in the library at30%, a CDRH3 comprising about 15 amino acids in length is present in thelibrary at 20%, and a CDRH3 comprising about 12 amino acids in length ispresent in the library at 10%.

Libraries as described herein encoding for a VHH antibody comprisevariant CDR sequences that are shuffled to generate a library with atheoretical diversity of at least or about 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹,10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than 10²⁰sequences. In some instances, the library has a final library diversityof at least or about 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵,10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, 10²⁰, or more than 10²⁰ sequences.

Provided herein are adenosine A2B receptor binding libraries encodingfor an immunoglobulin. In some instances, the adenosine A2B receptorimmunoglobulin is an antibody. In some instances, the adenosine A2Breceptor immunoglobulin is a VHH antibody. In some instances, theadenosine A2B receptor immunoglobulin comprises a binding affinity(e.g., K_(D)) to adenosine A2A receptor of less than 1 nM, less than 1.2nM, less than 2 nM, less than 5 nM, less than 10 nM, less than 11 nm,less than 13.5 nM, less than 15 nM, less than 20 nM, less than 25 nM, orless than 30 nM. In some instances, the adenosine A2B receptorimmunoglobulin comprises a K_(D) of less than 1 nM. In some instances,the adenosine A2B receptor immunoglobulin comprises a K_(D) of less than1.2 nM. In some instances, the adenosine A2B receptor immunoglobulincomprises a K_(D) of less than 2 nM. In some instances, the adenosineA2B receptor immunoglobulin comprises a K_(D) of less than 5 nM. In someinstances, the adenosine A2B receptor immunoglobulin comprises a K_(D)of less than 10 nM. In some instances, the adenosine A2B receptorimmunoglobulin comprises a K_(D) of less than 13.5 nM. In someinstances, the adenosine A2B receptor immunoglobulin comprises a K_(D)of less than 15 nM. In some instances, the adenosine A2B receptorimmunoglobulin comprises a K_(D) of less than 20 nM. In some instances,the adenosine A2B receptor immunoglobulin comprises a K_(D) of less than25 nM. In some instances, the adenosine A2B receptor immunoglobulincomprises a K_(D) of less than 30 nM.

In some instances, the adenosine A2B receptor immunoglobulin is anadenosine A2B receptor agonist. In some instances, the adenosine A2Breceptor immunoglobulin is an adenosine A2B receptor antagonist. In someinstances, the adenosine A2B receptor immunoglobulin is an adenosine A2Breceptor allosteric modulator. In some instances, the allostericmodulator is a negative allosteric modulator. In some instances, theallosteric modulator is a positive allosteric modulator. In someinstances, the adenosine A2B receptor immunoglobulin results inagonistic, antagonistic, or allosteric effects at a concentration of atleast or about 1 nM, 2 nM, 4 nM, 6 nM, 8 nM, 10 nM, 20 nM, 30 nM, 40 nM,50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 120 nM, 140 nM, 160 nM, 180nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1000nM, or more than 1000 nM. In some instances, the adenosine A2B receptorimmunoglobulin is a negative allosteric modulator. In some instances,the adenosine A2B receptor immunoglobulin is a negative allostericmodulator at a concentration of at least or about 0.001, 0.005, 0.01,0.05, 0.1, 0.5, 1 nM, 2 nM, 4 nM, 6 nM, 8 nM, 10 nM, 20 nM, 30 nM, 40nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, or more than 100 nM. Insome instances, the adenosine A2B receptor immunoglobulin is a negativeallosteric modulator at a concentration in a range of about 0.001 toabout 100, 0.01 to about 90, about 0.1 to about 80, 1 to about 50, about10 to about 40 nM, or about 1 to about 10 nM. In some instances, theadenosine A2B receptor immunoglobulin comprises an EC50 or IC50 of atleast or about 0.001, 0.0025, 0.005, 0.01, 0.025, 0.05, 0.06, 0.07,0.08, 0.9, 0.1, 0.5, 1, 2, 3, 4, 5, 6, or more than 6 nM. In someinstances, the adenosine A2B receptor immunoglobulin comprises an EC50or IC50 of at least or about 1 nM, 2 nM, 4 nM, 6 nM, 8 nM, 10 nM, 20 nM,30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, or more than100 nM.

Adenosine A2B receptor immunoglobulins as described herein may compriseimproved properties. In some instances, the adenosine A2B receptorimmunoglobulins are monomeric. In some instances, the adenosine A2Breceptor immunoglobulins are not prone to aggregation. In someinstances, at least or about 70%, 75%, 80%, 85%, 90%, 95%, or 99% of theadenosine A2B receptor immunoglobulins are monomeric. In some instances,the adenosine A2B receptor immunoglobulins are thermostable. In someinstances, the adenosine A2B receptor immunoglobulins result in reducednon-specific binding.

Following synthesis of adenosine A2B receptor binding librariescomprising nucleic acids encoding scaffolds comprising adenosine A2Breceptor binding domains, libraries may be used for screening andanalysis. For example, libraries are assayed for library displayabilityand panning. In some instances, displayability is assayed using aselectable tag. Exemplary tags include, but are not limited to, aradioactive label, a fluorescent label, an enzyme, a chemiluminescenttag, a colorimetric tag, an affinity tag or other labels or tags thatare known in the art. In some instances, the tag is histidine,polyhistidine, myc, hemagglutinin (HA), or FLAG. The adenosine A2Breceptor binding libraries may comprise nucleic acids encoding scaffoldscomprising adenosine A2B receptor binding domains with multiple tagssuch as GFP, FLAG, and Lucy as well as a DNA barcode. In some instances,libraries are assayed by sequencing using various methods including, butnot limited to, single-molecule real-time (SMRT) sequencing, Polonysequencing, sequencing by ligation, reversible terminator sequencing,proton detection sequencing, ion semiconductor sequencing, nanoporesequencing, electronic sequencing, pyrosequencing, Maxam-Gilbertsequencing, chain termination (e.g., Sanger) sequencing, +S sequencing,or sequencing by synthesis.

Expression Systems

Provided herein are libraries comprising nucleic acids encoding forscaffolds comprising adenosine A2A receptor binding domains, adenosineA2B receptor binding domains, or a combination thereof, wherein thelibraries have improved specificity, stability, expression, folding, ordownstream activity. In some instances, libraries described herein areused for screening and analysis.

Provided herein are libraries comprising nucleic acids encoding forscaffolds comprising adenosine A2A receptor binding domains, adenosineA2B receptor binding domains, or a combination thereof, wherein thenucleic acid libraries are used for screening and analysis. In someinstances, screening and analysis comprises in vitro, in vivo, or exvivo assays. Cells for screening include primary cells taken from livingsubjects or cell lines. Cells may be from prokaryotes (e.g., bacteriaand fungi) or eukaryotes (e.g., animals and plants). Exemplary animalcells include, without limitation, those from a mouse, rabbit, primate,and insect. In some instances, cells for screening include a cell lineincluding, but not limited to, Chinese Hamster Ovary (CHO) cell line,human embryonic kidney (HEK) cell line, or baby hamster kidney (BHK)cell line. In some instances, nucleic acid libraries described hereinmay also be delivered to a multicellular organism. Exemplarymulticellular organisms include, without limitation, a plant, a mouse,rabbit, primate, and insect.

Nucleic acid libraries or protein libraries encoded thereof describedherein may be screened for various pharmacological or pharmacokineticproperties. In some instances, the libraries are screened using in vitroassays, in vivo assays, or ex vivo assays. For example, in vitropharmacological or pharmacokinetic properties that are screened include,but are not limited to, binding affinity, binding specificity, andbinding avidity. Exemplary in vivo pharmacological or pharmacokineticproperties of libraries described herein that are screened include, butare not limited to, therapeutic efficacy, activity, preclinical toxicityproperties, clinical efficacy properties, clinical toxicity properties,immunogenicity, potency, and clinical safety properties.

Pharmacological or pharmacokinetic properties that may be screenedinclude, but are not limited to, cell binding affinity and cellactivity. For example, cell binding affinity assays or cell activityassays are performed to determine agonistic, antagonistic, or allostericeffects of libraries described herein. In some instances, the cellactivity assay is a cAMP assay. In some instances, libraries asdescribed herein are compared to cell binding or cell activity ofligands of adenosine A2A receptor, adenosine A2B receptor, or bothadenosine A2A receptor and adenosine A2B receptor.

Libraries as described herein may be screened in cell-based assays or innon-cell-based assays. Examples of non-cell-based assays include, butare not limited to, using viral particles, using in vitro translationproteins, and using protealiposomes with adenosine A2A receptor,adenosine A2B receptor, or both adenosine A2A receptor and adenosine A2Breceptor.

Nucleic acid libraries as described herein may be screened bysequencing. In some instances, next generation sequence is used todetermine sequence enrichment of adenosine A2A receptor bindingvariants, adenosine A2B receptor binding variants, or a combinationthereof. In some instances, V gene distribution, J gene distribution, Vgene family, CDR3 counts per length, or a combination thereof isdetermined. In some instances, clonal frequency, clonal accumulation,lineage accumulation, or a combination thereof is determined. In someinstances, number of sequences, sequences with VH clones, clones, clonesgreater than 1, clonotypes, clonotypes greater than 1, lineages,simpsons, or a combination thereof is determined. In some instances, apercentage of non-identical CDR3s is determined. For example, thepercentage of non-identical CDR3s is calculated as the number ofnon-identical CDR3s in a sample divided by the total number of sequencesthat had a CDR3 in the sample.

Provided herein are nucleic acid libraries, wherein the nucleic acidlibraries may be expressed in a vector. Expression vectors for insertingnucleic acid libraries disclosed herein may comprise eukaryotic orprokaryotic expression vectors. Exemplary expression vectors include,without limitation, mammalian expression vectors:pSF-CMV-NEO-NH2-PPT-3XFLAG, pSF-CMV-NEO-COOH-3XFLAG,pSF-CMV-PURO-NH2-GST-TEV, pSF-OXB20-COOH-TEV-FLAG(R)-6His, pCEP4pDEST27, pSF-CMV-Ub-KrYFP, pSF-CMV-FMDV-daGFP, pEF1a-mCherry-N1 Vector,pEF1a-tdTomato Vector, pSF-CMV-FMDV-Hygro, pSF-CMV-PGK-Puro,pMCP-tag(m), and pSF-CMV-PURO-NH2-CMYC; bacterial expression vectors:pSF-OXB20-BetaGal, pSF-OXB20-Fluc, pSF-OXB20, and pSF-Tac; plantexpression vectors: pRI 101-AN DNA and pCambia2301; and yeast expressionvectors: pTYB21 and pKLAC2, and insect vectors: pAc5.1/V5-His A andpDEST8. In some instances, the vector is pcDNA3 or pcDNA3.1.

Described herein are nucleic acid libraries that are expressed in avector to generate a construct comprising a scaffold comprisingsequences of adenosine A2A receptor binding domains, adenosine A2Breceptor domains, or a combination thereof. In some instances, a size ofthe construct varies. In some instances, the construct comprises atleast or about 500, 600, 700, 800, 900, 1000, 1100, 1300, 1400, 1500,1600, 1700, 1800, 2000, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800,4000, 4200, 4400, 4600, 4800, 5000, 6000, 7000, 8000, 9000, 10000, ormore than 10000 bases. In some instances, a the construct comprises arange of about 300 to 1,000, 300 to 2,000, 300 to 3,000, 300 to 4,000,300 to 5,000, 300 to 6,000, 300 to 7,000, 300 to 8,000, 300 to 9,000,300 to 10,000, 1,000 to 2,000, 1,000 to 3,000, 1,000 to 4,000, 1,000 to5,000, 1,000 to 6,000, 1,000 to 7,000, 1,000 to 8,000, 1,000 to 9,000,1,000 to 10,000, 2,000 to 3,000, 2,000 to 4,000, 2,000 to 5,000, 2,000to 6,000, 2,000 to 7,000, 2,000 to 8,000, 2,000 to 9,000, 2,000 to10,000, 3,000 to 4,000, 3,000 to 5,000, 3,000 to 6,000, 3,000 to 7,000,3,000 to 8,000, 3,000 to 9,000, 3,000 to 10,000, 4,000 to 5,000, 4,000to 6,000, 4,000 to 7,000, 4,000 to 8,000, 4,000 to 9,000, 4,000 to10,000, 5,000 to 6,000, 5,000 to 7,000, 5,000 to 8,000, 5,000 to 9,000,5,000 to 10,000, 6,000 to 7,000, 6,000 to 8,000, 6,000 to 9,000, 6,000to 10,000, 7,000 to 8,000, 7,000 to 9,000, 7,000 to 10,000, 8,000 to9,000, 8,000 to 10,000, or 9,000 to 10,000 bases.

Provided herein are libraries comprising nucleic acids encoding forscaffolds comprising adenosine A2A receptor binding domains, adenosineA2B receptor domains, or a combination thereof, wherein the nucleic acidlibraries are expressed in a cell. In some instances, the libraries aresynthesized to express a reporter gene. Exemplary reporter genesinclude, but are not limited to, acetohydroxyacid synthase (AHAS),alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase(GUS), chloramphenicol acetyltransferase (CAT), green fluorescentprotein (GFP), red fluorescent protein (RFP), yellow fluorescent protein(YFP), cyan fluorescent protein (CFP), cerulean fluorescent protein,citrine fluorescent protein, orange fluorescent protein, cherryfluorescent protein, turquoise fluorescent protein, blue fluorescentprotein, horseradish peroxidase (HRP), luciferase (Luc), nopalinesynthase (NOS), octopine synthase (OCS), luciferase, and derivativesthereof. Methods to determine modulation of a reporter gene are wellknown in the art, and include, but are not limited to, fluorometricmethods (e.g. fluorescence spectroscopy, Fluorescence Activated CellSorting (FACS), fluorescence microscopy), and antibiotic resistancedetermination.

Diseases and Disorders

Provided herein are adenosine A2A receptor binding libraries comprisingnucleic acids encoding for scaffolds comprising adenosine A2A receptorbinding domains that may have therapeutic effects. Also provided hereinare adenosine A2B receptor binding libraries comprising nucleic acidsencoding for scaffolds comprising adenosine A2B receptor binding domainsthat may have therapeutic effects. In some instances, the adenosine A2Areceptor binding libraries and adenosine A2B receptor libraries resultin protein when translated that is used to treat a disease or disorder.In some instances, the protein is an immunoglobulin. In some instances,the protein is a peptidomimetic. Exemplary diseases include, but are notlimited to, cancer, inflammatory diseases or disorders, a metabolicdisease or disorder, a cardiovascular disease or disorder, a respiratorydisease or disorder, pain, a digestive disease or disorder, areproductive disease or disorder, an endocrine disease or disorder, or aneurological disease or disorder. In some instances, an inhibitor ofadenosine A2A receptor, adenosine A2B receptor, or a combination thereofas described herein is used for treatment of a disease or disorder ofthe central nervous system, kidney, intestine, lung, hair, skin, bone,or cartilage. In some instances, an inhibitor of adenosine A2A receptor,adenosine A2B receptor, or a combination thereof as described herein isused for sleep regulation, angiogenesis, or modulation of the immunesystem.

In some instances, the A2AR immunoglobulins, A2BR immunoglobulins, or acombination thereof described herein are used for treating aneurological disease or disorder. In some instances, the neurologicaldisease or disorder is a neurodegenerative disease or disorder. In someinstances, the neurological disease or disorder is Parkinson's disease,Alzheimer's disease, or multiple sclerosis.

In some instances, the A2AR immunoglobulins, A2BR immunoglobulins, or acombination thereof described herein are used for treating cancer. Insome instances, the cancer is a solid cancer or a hematologic cancer. Insome instances, the A2AR immunoglobulins, A2BR immunoglobulins, or acombination thereof described herein are used as a monotherapy fortreating cancer. In some instances, the A2AR immunoglobulins, A2BRimmunoglobulins, or a combination thereof described herein are used incombination with other therapeutic agents for treating cancer. In someembodiments, the cancer is lung, colorectal, or prostate cancer. In someinstances, the A2AR immunoglobulins, A2BR immunoglobulins, or acombination thereof described herein enhance tumor vaccines, checkpointblockade and adoptive T cell therapy. In some instances, the A2ARimmunoglobulin, A2BR immunoglobulins, or a combination thereof targetsimmune cells and blocks immunosuppression in order to treat cancer.

In some instances, the A2AR immunoglobulins, A2BR immunoglobulins, or acombination thereof described herein reduce tumor size by at least orabout 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or more than 95%. In some instances, the A2ARimmunoglobulins, A2BR immunoglobulins, or a combination thereofdescribed herein reduce tumor size by at least or about 10%, 15%, 20%,25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, ormore than 95% compared to a comparator antibody (e.g., pembrolizumab ornivolumab) or a control. In some instances, the control is no treatmentor placebo.

In some instances, the A2AR immunoglobulins, A2BR immunoglobulins, or acombination thereof described herein increase cell number of cells ofthe lymphoid or myeloid compartment. In some instances, the A2ARimmunoglobulins, A2BR immunoglobulins, or a combination thereofdescribed herein increase tumor infiltrating lymphocytes (TIL) CD45+cells, total T-cells, CD4+ cells, CD8+ cells, regulatory T-cells(Tregs), M1 tumor associated macrophages (TAM), or combinations thereofby at least or about 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%. In some instances,the A2AR immunoglobulins, A2BR immunoglobulins, or a combination thereofdescribed herein increase tumor infiltrating lymphocytes (TIL) CD45+cells, total T-cells, CD4+ cells, CD8+ cells, regulatory T-cells(Tregs), M1 tumor associated macrophages (TAM), or combinations thereofby at least or about 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95% compared to acomparator antibody (e.g., pembrolizumab or nivolumab) or a control. Insome instances, the control is no treatment or placebo.

In some instances, the A2AR immunoglobulins, A2BR immunoglobulins, or acombination thereof described herein increase cytokine expression. Insome embodiments, the cytokine is interferon gamma, interleukin 2,interleukin 4, interleukin 6, interleukin 8, interleukin 10, or TNFalpha. In some embodiments, the cytokine is interleukin 1β, interleukin1Ra, GM-CSF, interleukin 2, interleukin 7, interleukin 15, interleukin6, interleukin 6, interleukin 10, interferon gamma, or TNF alpha. Insome instances, the A2AR immunoglobulins, A2BR immunoglobulins, or acombination thereof described herein increase cytokine expression by atleast or about 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or more than 95%. In some instances, theA2AR immunoglobulins, A2BR immunoglobulins, or a combination thereofdescribed herein increase cytokine expression by at least or about 10%,15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or more than 95% compared to a comparator antibody (e.g.,pembrolizumab or nivolumab) or a control. In some instances, the controlis no treatment or placebo.

In some instances, the subject is a mammal. In some instances, thesubject is a mouse, rabbit, dog, or human. Subjects treated by methodsdescribed herein may be infants, adults, or children. Pharmaceuticalcompositions comprising antibodies or antibody fragments as describedherein may be administered intravenously or subcutaneously.

Variant Libraries

Codon Variation

Variant nucleic acid libraries described herein may comprise a pluralityof nucleic acids, wherein each nucleic acid encodes for a variant codonsequence compared to a reference nucleic acid sequence. In someinstances, each nucleic acid of a first nucleic acid population containsa variant at a single variant site. In some instances, the first nucleicacid population contains a plurality of variants at a single variantsite such that the first nucleic acid population contains more than onevariant at the same variant site. The first nucleic acid population maycomprise nucleic acids collectively encoding multiple codon variants atthe same variant site. The first nucleic acid population may comprisenucleic acids collectively encoding up to 19 or more codons at the sameposition. The first nucleic acid population may comprise nucleic acidscollectively encoding up to 60 variant triplets at the same position, orthe first nucleic acid population may comprise nucleic acidscollectively encoding up to 61 different triplets of codons at the sameposition. Each variant may encode for a codon that results in adifferent amino acid during translation. Table 2 provides a listing ofeach codon possible (and the representative amino acid) for a variantsite.

TABLE 2 List of codons and amino acids One Three letter letterAmino Acids code code Codons Alanine A Ala GCA GCC GCG GCT Cysteine CCys TGC TGT Aspartic acid D Asp GAC GAT Glutamic acid E Glu GAA GAGPhenylalanine F Phe TTC TTT Glycine G Gly GGA GGC GGG GGT Histidine HHis CAC CAT Isoleucine  I Iso ATA ATC ATT Lysine K Lys AAA AAG Leucine LLeu TTA TTG CTA CTC CTG CTT Methionine  M Met ATG Asparagine  N Asn AACAAT Proline P Pro CCA CCC CCG CCT Glutamine Q Gln CAA CAG Arginine R ArgAGA AGG CGA CGC CGG CGT Serine S Ser AGC AGT TCA TCC TCG TCT Threonine TThr ACA ACC ACG ACT Valine V Val GTA GTC GTG GTT Tryptophan  W Trp TGGTyrosine Y Tyr TAC TAT

A nucleic acid population may comprise varied nucleic acids collectivelyencoding up to 20 codon variations at multiple positions. In such cases,each nucleic acid in the population comprises variation for codons atmore than one position in the same nucleic acid. In some instances, eachnucleic acid in the population comprises variation for codons at 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or morecodons in a single nucleic acid. In some instances, each variant longnucleic acid comprises variation for codons at 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30 or more codons in a single long nucleic acid. In someinstances, the variant nucleic acid population comprises variation forcodons at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more codons in asingle nucleic acid. In some instances, the variant nucleic acidpopulation comprises variation for codons in at least about 10, 20, 30,40, 50, 60, 70, 80, 90, 100 or more codons in a single long nucleicacid.

Highly Parallel Nucleic Acid Synthesis

Provided herein is a platform approach utilizing miniaturization,parallelization, and vertical integration of the end-to-end process frompolynucleotide synthesis to gene assembly within nanowells on silicon tocreate a revolutionary synthesis platform. Devices described hereinprovide, with the same footprint as a 96-well plate, a silicon synthesisplatform is capable of increasing throughput by a factor of up to 1,000or more compared to traditional synthesis methods, with production of upto approximately 1,000,000 or more polynucleotides, or 10,000 or moregenes in a single highly-parallelized run.

With the advent of next-generation sequencing, high resolution genomicdata has become an important factor for studies that delve into thebiological roles of various genes in both normal biology and diseasepathogenesis. At the core of this research is the central dogma ofmolecular biology and the concept of “residue-by-residue transfer ofsequential information.” Genomic information encoded in the DNA istranscribed into a message that is then translated into the protein thatis the active product within a given biological pathway.

Another exciting area of study is on the discovery, development andmanufacturing of therapeutic molecules focused on a highly-specificcellular target. High diversity DNA sequence libraries are at the coreof development pipelines for targeted therapeutics. Gene mutants areused to express proteins in a design, build, and test proteinengineering cycle that ideally culminates in an optimized gene for highexpression of a protein with high affinity for its therapeutic target.As an example, consider the binding pocket of a receptor. The ability totest all sequence permutations of all residues within the binding pocketsimultaneously will allow for a thorough exploration, increasing chancesof success. Saturation mutagenesis, in which a researcher attempts togenerate all possible mutations at a specific site within the receptor,represents one approach to this development challenge. Though costly andtime and labor-intensive, it enables each variant to be introduced intoeach position. In contrast, combinatorial mutagenesis, where a fewselected positions or short stretch of DNA may be modified extensively,generates an incomplete repertoire of variants with biasedrepresentation.

To accelerate the drug development pipeline, a library with the desiredvariants available at the intended frequency in the right positionavailable for testing—in other words, a precision library, enablesreduced costs as well as turnaround time for screening. Provided hereinare methods for synthesizing nucleic acid synthetic variant librarieswhich provide for precise introduction of each intended variant at thedesired frequency. To the end user, this translates to the ability tonot only thoroughly sample sequence space but also be able to querythese hypotheses in an efficient manner, reducing cost and screeningtime. Genome-wide editing can elucidate important pathways, librarieswhere each variant and sequence permutation can be tested for optimalfunctionality, and thousands of genes can be used to reconstruct entirepathways and genomes to re-engineer biological systems for drugdiscovery.

In a first example, a drug itself can be optimized using methodsdescribed herein. For example, to improve a specified function of anantibody, a variant polynucleotide library encoding for a portion of theantibody is designed and synthesized. A variant nucleic acid library forthe antibody can then be generated by processes described herein (e.g.,PCR mutagenesis followed by insertion into a vector). The antibody isthen expressed in a production cell line and screened for enhancedactivity. Example screens include examining modulation in bindingaffinity to an antigen, stability, or effector function (e.g., ADCC,complement, or apoptosis). Exemplary regions to optimize the antibodyinclude, without limitation, the Fc region, Fab region, variable regionof the Fab region, constant region of the Fab region, variable domain ofthe heavy chain or light chain (V_(H) or V_(L)), and specificcomplementarity-determining regions (CDRs) of V_(H) or V_(L).

Nucleic acid libraries synthesized by methods described herein may beexpressed in various cells associated with a disease state. Cellsassociated with a disease state include cell lines, tissue samples,primary cells from a subject, cultured cells expanded from a subject, orcells in a model system. Exemplary model systems include, withoutlimitation, plant and animal models of a disease state.

To identify a variant molecule associated with prevention, reduction ortreatment of a disease state, a variant nucleic acid library describedherein is expressed in a cell associated with a disease state, or one inwhich a cell a disease state can be induced. In some instances, an agentis used to induce a disease state in cells. Exemplary tools for diseasestate induction include, without limitation, a Cre/Lox recombinationsystem, LPS inflammation induction, and streptozotocin to inducehypoglycemia. The cells associated with a disease state may be cellsfrom a model system or cultured cells, as well as cells from a subjecthaving a particular disease condition. Exemplary disease conditionsinclude a bacterial, fungal, viral, autoimmune, or proliferativedisorder (e.g., cancer). In some instances, the variant nucleic acidlibrary is expressed in the model system, cell line, or primary cellsderived from a subject, and screened for changes in at least onecellular activity. Exemplary cellular activities include, withoutlimitation, proliferation, cycle progression, cell death, adhesion,migration, reproduction, cell signaling, energy production, oxygenutilization, metabolic activity, and aging, response to free radicaldamage, or any combination thereof

Substrates

Devices used as a surface for polynucleotide synthesis may be in theform of substrates which include, without limitation, homogenous arraysurfaces, patterned array surfaces, channels, beads, gels, and the like.Provided herein are substrates comprising a plurality of clusters,wherein each cluster comprises a plurality of loci that support theattachment and synthesis of polynucleotides. In some instances,substrates comprise a homogenous array surface. For example, thehomogenous array surface is a homogenous plate. The term “locus” as usedherein refers to a discrete region on a structure which provides supportfor polynucleotides encoding for a single predetermined sequence toextend from the surface. In some instances, a locus is on a twodimensional surface, e.g., a substantially planar surface. In someinstances, a locus is on a three-dimensional surface, e.g., a well,microwell, channel, or post. In some instances, a surface of a locuscomprises a material that is actively functionalized to attach to atleast one nucleotide for polynucleotide synthesis, or preferably, apopulation of identical nucleotides for synthesis of a population ofpolynucleotides. In some instances, polynucleotide refers to apopulation of polynucleotides encoding for the same nucleic acidsequence. In some cases, a surface of a substrate is inclusive of one ora plurality of surfaces of a substrate. The average error rates forpolynucleotides synthesized within a library described here using thesystems and methods provided are often less than 1 in 1000, less thanabout 1 in 2000, less than about 1 in 3000 or less often without errorcorrection.

Provided herein are surfaces that support the parallel synthesis of aplurality of polynucleotides having different predetermined sequences ataddressable locations on a common support. In some instances, asubstrate provides support for the synthesis of more than 50, 100, 200,400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2,000; 5,000; 10,000;20,000; 50,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000;700,000; 800,000; 900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000;1,800,000; 2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000;4,500,000; 5,000,000; 10,000,000 or more non-identical polynucleotides.In some cases, the surfaces provide support for the synthesis of morethan 50, 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2,000;5,000; 10,000; 20,000; 50,000; 100,000; 200,000; 300,000; 400,000;500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,200,000;1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000; 3,000,000;3,500,000; 4,000,000; 4,500,000; 5,000,000; 10,000,000 or morepolynucleotides encoding for distinct sequences. In some instances, atleast a portion of the polynucleotides have an identical sequence or areconfigured to be synthesized with an identical sequence. In someinstances, the substrate provides a surface environment for the growthof polynucleotides having at least 80, 90, 100, 120, 150, 175, 200, 225,250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 bases or more.

Provided herein are methods for polynucleotide synthesis on distinctloci of a substrate, wherein each locus supports the synthesis of apopulation of polynucleotides. In some cases, each locus supports thesynthesis of a population of polynucleotides having a different sequencethan a population of polynucleotides grown on another locus. In someinstances, each polynucleotide sequence is synthesized with 1, 2, 3, 4,5, 6, 7, 8, 9 or more redundancy across different loci within the samecluster of loci on a surface for polynucleotide synthesis. In someinstances, the loci of a substrate are located within a plurality ofclusters. In some instances, a substrate comprises at least 10, 500,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000,12000, 13000, 14000, 15000, 20000, 30000, 40000, 50000 or more clusters.In some instances, a substrate comprises more than 2,000; 5,000; 10,000;100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000;900,000; 1,000,000; 1,100,000; 1,200,000; 1,300,000; 1,400,000;1,500,000; 1,600,000; 1,700,000; 1,800,000; 1,900,000; 2,000,000;300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000;1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000;2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; or10,000,000 or more distinct loci. In some instances, a substratecomprises about 10,000 distinct loci. The amount of loci within a singlecluster is varied in different instances. In some cases, each clusterincludes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 120, 130, 150, 200, 300, 400, 500 or more loci. In some instances,each cluster includes about 50-500 loci. In some instances, each clusterincludes about 100-200 loci. In some instances, each cluster includesabout 100-150 loci. In some instances, each cluster includes about 109,121, 130 or 137 loci. In some instances, each cluster includes about 19,20, 61, 64 or more loci. Alternatively or in combination, polynucleotidesynthesis occurs on a homogenous array surface.

In some instances, the number of distinct polynucleotides synthesized ona substrate is dependent on the number of distinct loci available in thesubstrate. In some instances, the density of loci within a cluster orsurface of a substrate is at least or about 1, 10, 25, 50, 65, 75, 100,130, 150, 175, 200, 300, 400, 500, 1,000 or more loci per mm². In somecases, a substrate comprises 10-500, 25-400, 50-500, 100-500, 150-500,10-250, 50-250, 10-200, or 50-200 mm². In some instances, the distancebetween the centers of two adjacent loci within a cluster or surface isfrom about 10-500, from about 10-200, or from about 10-100 um. In someinstances, the distance between two centers of adjacent loci is greaterthan about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 um. In someinstances, the distance between the centers of two adjacent loci is lessthan about 200, 150, 100, 80, 70, 60, 50, 40, 30, 20 or 10 um. In someinstances, each locus has a width of about 0.5, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 um. In some cases, eachlocus has a width of about 0.5-100, 0.5-50, 10-75, or 0.5-50 um.

In some instances, the density of clusters within a substrate is atleast or about 1 cluster per 100 mm², 1 cluster per 10 mm², 1 clusterper 5 mm², 1 cluster per 4 mm², 1 cluster per 3 mm², 1 cluster per 2mm², 1 cluster per 1 mm², 2 clusters per 1 mm², 3 clusters per 1 mm², 4clusters per 1 mm², 5 clusters per 1 mm², 10 clusters per 1 mm², 50clusters per 1 mm² or more. In some instances, a substrate comprisesfrom about 1 cluster per 10 mm² to about 10 clusters per 1 mm². In someinstances, the distance between the centers of two adjacent clusters isat least or about 50, 100, 200, 500, 1000, 2000, or 5000 um. In somecases, the distance between the centers of two adjacent clusters isbetween about 50-100, 50-200, 50-300, 50-500, and 100-2000 um. In somecases, the distance between the centers of two adjacent clusters isbetween about 0.05-50, 0.05-10, 0.05-5, 0.05-4, 0.05-3, 0.05-2, 0.1-10,0.2-10, 0.3-10, 0.4-10, 0.5-10, 0.5-5, or 0.5-2 mm. In some cases, eachcluster has a cross section of about 0.5 to about 2, about 0.5 to about1, or about 1 to about 2 mm. In some cases, each cluster has a crosssection of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9 or 2 mm. In some cases, each cluster has an interiorcross section of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.15, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 mm.

In some instances, a substrate is about the size of a standard 96 wellplate, for example between about 100 and about 200 mm by between about50 and about 150 mm. In some instances, a substrate has a diameter lessthan or equal to about 1000, 500, 450, 400, 300, 250, 200, 150, 100 or50 mm. In some instances, the diameter of a substrate is between about25-1000, 25-800, 25-600, 25-500, 25-400, 25-300, or 25-200 mm. In someinstances, a substrate has a planar surface area of at least about 100;200; 500; 1,000; 2,000; 5,000; 10,000; 12,000; 15,000; 20,000; 30,000;40,000; 50,000 mm² or more. In some instances, the thickness of asubstrate is between about 50-2000, 50-1000, 100-1000, 200-1000, or250-1000 mm.

Surface Materials

Substrates, devices, and reactors provided herein are fabricated fromany variety of materials suitable for the methods, compositions, andsystems described herein. In certain instances, substrate materials arefabricated to exhibit a low level of nucleotide binding. In someinstances, substrate materials are modified to generate distinctsurfaces that exhibit a high level of nucleotide binding. In someinstances, substrate materials are transparent to visible and/or UVlight. In some instances, substrate materials are sufficientlyconductive, e.g., are able to form uniform electric fields across all ora portion of a substrate. In some instances, conductive materials areconnected to an electric ground. In some instances, the substrate isheat conductive or insulated. In some instances, the materials arechemical resistant and heat resistant to support chemical or biochemicalreactions, for example polynucleotide synthesis reaction processes. Insome instances, a substrate comprises flexible materials. For flexiblematerials, materials can include, without limitation: nylon, bothmodified and unmodified, nitrocellulose, polypropylene, and the like. Insome instances, a substrate comprises rigid materials. For rigidmaterials, materials can include, without limitation: glass; fusesilica; silicon, plastics (for example polytetrafluoroethylene,polypropylene, polystyrene, polycarbonate, and blends thereof, and thelike); metals (for example, gold, platinum, and the like). Thesubstrate, solid support or reactors can be fabricated from a materialselected from the group consisting of silicon, polystyrene, agarose,dextran, cellulosic polymers, polyacrylamides, polydimethylsiloxane(PDMS), and glass. The substrates/solid supports or the microstructures,reactors therein may be manufactured with a combination of materialslisted herein or any other suitable material known in the art.

Surface Architecture

Provided herein are substrates for the methods, compositions, andsystems described herein, wherein the substrates have a surfacearchitecture suitable for the methods, compositions, and systemsdescribed herein. In some instances, a substrate comprises raised and/orlowered features. One benefit of having such features is an increase insurface area to support polynucleotide synthesis. In some instances, asubstrate having raised and/or lowered features is referred to as athree-dimensional substrate. In some cases, a three-dimensionalsubstrate comprises one or more channels. In some cases, one or moreloci comprise a channel. In some cases, the channels are accessible toreagent deposition via a deposition device such as a material depositiondevice. In some cases, reagents and/or fluids collect in a larger wellin fluid communication one or more channels. For example, a substratecomprises a plurality of channels corresponding to a plurality of lociwith a cluster, and the plurality of channels are in fluid communicationwith one well of the cluster. In some methods, a library ofpolynucleotides is synthesized in a plurality of loci of a cluster.

Provided herein are substrates for the methods, compositions, andsystems described herein, wherein the substrates are configured forpolynucleotide synthesis. In some instances, the structure is configuredto allow for controlled flow and mass transfer paths for polynucleotidesynthesis on a surface. In some instances, the configuration of asubstrate allows for the controlled and even distribution of masstransfer paths, chemical exposure times, and/or wash efficacy duringpolynucleotide synthesis. In some instances, the configuration of asubstrate allows for increased sweep efficiency, for example byproviding sufficient volume for a growing polynucleotide such that theexcluded volume by the growing polynucleotide does not take up more than50, 45, 40, 35, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2, 1%, or less of the initially available volume that is available orsuitable for growing the polynucleotide. In some instances, athree-dimensional structure allows for managed flow of fluid to allowfor the rapid exchange of chemical exposure.

Provided herein are substrates for the methods, compositions, andsystems described herein, wherein the substrates comprise structuressuitable for the methods, compositions, and systems described herein. Insome instances, segregation is achieved by physical structure. In someinstances, segregation is achieved by differential functionalization ofthe surface generating active and passive regions for polynucleotidesynthesis. In some instances, differential functionalization is achievedby alternating the hydrophobicity across the substrate surface, therebycreating water contact angle effects that cause beading or wetting ofthe deposited reagents. Employing larger structures can decreasesplashing and cross-contamination of distinct polynucleotide synthesislocations with reagents of the neighboring spots. In some cases, adevice, such as a material deposition device, is used to depositreagents to distinct polynucleotide synthesis locations. Substrateshaving three-dimensional features are configured in a manner that allowsfor the synthesis of a large number of polynucleotides (e.g., more thanabout 10,000) with a low error rate (e.g., less than about 1:500,1:1000, 1:1500, 1:2,000, 1:3,000, 1:5,000, or 1:10,000). In some cases,a substrate comprises features with a density of about or greater thanabout 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 300, 400 or 500 features per mm².

A well of a substrate may have the same or different width, height,and/or volume as another well of the substrate. A channel of a substratemay have the same or different width, height, and/or volume as anotherchannel of the substrate. In some instances, the diameter of a clusteror the diameter of a well comprising a cluster, or both, is betweenabout 0.05-50, 0.05-10, 0.05-5, 0.05-4, 0.05-3, 0.05-2, 0.05-1,0.05-0.5, 0.05-0.1, 0.1-10, 0.2-10, 0.3-10, 0.4-10, 0.5-10, 0.5-5, or0.5-2 mm. In some instances, the diameter of a cluster or well or bothis less than or about 5, 4, 3, 2, 1, 0.5, 0.1, 0.09, 0.08, 0.07, 0.06,or 0.05 mm. In some instances, the diameter of a cluster or well or bothis between about 1.0 and 1.3 mm. In some instances, the diameter of acluster or well, or both is about 1.150 mm. In some instances, thediameter of a cluster or well, or both is about 0.08 mm. The diameter ofa cluster refers to clusters within a two-dimensional orthree-dimensional substrate.

In some instances, the height of a well is from about 20-1000, 50-1000,100-1000, 200-1000, 300-1000, 400-1000, or 500-1000 um. In some cases,the height of a well is less than about 1000, 900, 800, 700, or 600 um.

In some instances, a substrate comprises a plurality of channelscorresponding to a plurality of loci within a cluster, wherein theheight or depth of a channel is 5-500, 5-400, 5-300, 5-200, 5-100, 5-50,or 10-50 um. In some cases, the height of a channel is less than 100,80, 60, 40, or 20 um.

In some instances, the diameter of a channel, locus (e.g., in asubstantially planar substrate) or both channel and locus (e.g., in athree-dimensional substrate wherein a locus corresponds to a channel) isfrom about 1-1000, 1-500, 1-200, 1-100, 5-100, or 10-100 um, forexample, about 90, 80, 70, 60, 50, 40, 30, 20 or 10 um. In someinstances, the diameter of a channel, locus, or both channel and locusis less than about 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 um. In someinstances, the distance between the center of two adjacent channels,loci, or channels and loci is from about 1-500, 1-200, 1-100, 5-200,5-100, 5-50, or 5-30, for example, about 20 um.

Surface Modifications

Provided herein are methods for polynucleotide synthesis on a surface,wherein the surface comprises various surface modifications. In someinstances, the surface modifications are employed for the chemicaland/or physical alteration of a surface by an additive or subtractiveprocess to change one or more chemical and/or physical properties of asubstrate surface or a selected site or region of a substrate surface.For example, surface modifications include, without limitation, (1)changing the wetting properties of a surface, (2) functionalizing asurface, i.e., providing, modifying or substituting surface functionalgroups, (3) defunctionalizing a surface, i.e., removing surfacefunctional groups, (4) otherwise altering the chemical composition of asurface, e.g., through etching, (5) increasing or decreasing surfaceroughness, (6) providing a coating on a surface, e.g., a coating thatexhibits wetting properties that are different from the wettingproperties of the surface, and/or (7) depositing particulates on asurface.

In some cases, the addition of a chemical layer on top of a surface(referred to as adhesion promoter) facilitates structured patterning ofloci on a surface of a substrate. Exemplary surfaces for application ofadhesion promotion include, without limitation, glass, silicon, silicondioxide and silicon nitride. In some cases, the adhesion promoter is achemical with a high surface energy. In some instances, a secondchemical layer is deposited on a surface of a substrate. In some cases,the second chemical layer has a low surface energy. In some cases,surface energy of a chemical layer coated on a surface supportslocalization of droplets on the surface. Depending on the patterningarrangement selected, the proximity of loci and/or area of fluid contactat the loci are alterable.

In some instances, a substrate surface, or resolved loci, onto whichnucleic acids or other moieties are deposited, e.g., for polynucleotidesynthesis, are smooth or substantially planar (e.g., two-dimensional) orhave irregularities, such as raised or lowered features (e.g.,three-dimensional features). In some instances, a substrate surface ismodified with one or more different layers of compounds. Suchmodification layers of interest include, without limitation, inorganicand organic layers such as metals, metal oxides, polymers, small organicmolecules and the like.

In some instances, resolved loci of a substrate are functionalized withone or more moieties that increase and/or decrease surface energy. Insome cases, a moiety is chemically inert. In some cases, a moiety isconfigured to support a desired chemical reaction, for example, one ormore processes in a polynucleotide synthesis reaction. The surfaceenergy, or hydrophobicity, of a surface is a factor for determining theaffinity of a nucleotide to attach onto the surface. In some instances,a method for substrate functionalization comprises: (a) providing asubstrate having a surface that comprises silicon dioxide; and (b)silanizing the surface using, a suitable silanizing agent describedherein or otherwise known in the art, for example, an organofunctionalalkoxysilane molecule. Methods and functionalizing agents are describedin U.S. Pat. No. 5,474,796, which is herein incorporated by reference inits entirety.

In some instances, a substrate surface is functionalized by contact witha derivatizing composition that contains a mixture of silanes, underreaction conditions effective to couple the silanes to the substratesurface, typically via reactive hydrophilic moieties present on thesubstrate surface. Silanization generally covers a surface throughself-assembly with organofunctional alkoxysilane molecules. A variety ofsiloxane functionalizing reagents can further be used as currently knownin the art, e.g., for lowering or increasing surface energy. Theorganofunctional alkoxysilanes are classified according to their organicfunctions.

Polynucleotide Synthesis

Methods of the current disclosure for polynucleotide synthesis mayinclude processes involving phosphoramidite chemistry. In someinstances, polynucleotide synthesis comprises coupling a base withphosphoramidite. Polynucleotide synthesis may comprise coupling a baseby deposition of phosphoramidite under coupling conditions, wherein thesame base is optionally deposited with phosphoramidite more than once,i.e., double coupling. Polynucleotide synthesis may comprise capping ofunreacted sites. In some instances, capping is optional. Polynucleotidesynthesis may also comprise oxidation or an oxidation step or oxidationsteps. Polynucleotide synthesis may comprise deblocking, detritylation,and sulfurization. In some instances, polynucleotide synthesis compriseseither oxidation or sulfurization. In some instances, between one oreach step during a polynucleotide synthesis reaction, the device iswashed, for example, using tetrazole or acetonitrile. Time frames forany one step in a phosphoramidite synthesis method may be less thanabout 2 min, 1 min, 50 sec, 40 sec, 30 sec, 20 sec and 10 sec.

Polynucleotide synthesis using a phosphoramidite method may comprise asubsequent addition of a phosphoramidite building block (e.g.,nucleoside phosphoramidite) to a growing polynucleotide chain for theformation of a phosphite triester linkage. Phosphoramiditepolynucleotide synthesis proceeds in the 3′ to 5′ direction.Phosphoramidite polynucleotide synthesis allows for the controlledaddition of one nucleotide to a growing nucleic acid chain per synthesiscycle. In some instances, each synthesis cycle comprises a couplingstep. Phosphoramidite coupling involves the formation of a phosphitetriester linkage between an activated nucleoside phosphoramidite and anucleoside bound to the substrate, for example, via a linker. In someinstances, the nucleoside phosphoramidite is provided to the deviceactivated. In some instances, the nucleoside phosphoramidite is providedto the device with an activator. In some instances, nucleosidephosphoramidites are provided to the device in a 1.5, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50,60, 70, 80, 90, 100-fold excess or more over the substrate-boundnucleosides. In some instances, the addition of nucleosidephosphoramidite is performed in an anhydrous environment, for example,in anhydrous acetonitrile. Following addition of a nucleosidephosphoramidite, the device is optionally washed. In some instances, thecoupling step is repeated one or more additional times, optionally witha wash step between nucleoside phosphoramidite additions to thesubstrate. In some instances, a polynucleotide synthesis method usedherein comprises 1, 2, 3 or more sequential coupling steps. Prior tocoupling, in many cases, the nucleoside bound to the device isde-protected by removal of a protecting group, where the protectinggroup functions to prevent polymerization. A common protecting group is4,4′-dimethoxytrityl (DMT).

Following coupling, phosphoramidite polynucleotide synthesis methodsoptionally comprise a capping step. In a capping step, the growingpolynucleotide is treated with a capping agent. A capping step is usefulto block unreacted substrate-bound 5′-OH groups after coupling fromfurther chain elongation, preventing the formation of polynucleotideswith internal base deletions. Further, phosphoramidites activated with1H-tetrazole may react, to a small extent, with the O6 position ofguanosine. Without being bound by theory, upon oxidation with I₂/water,this side product, possibly via O6-N7 migration, may undergodepurination. The apurinic sites may end up being cleaved in the courseof the final deprotection of the polynucleotide thus reducing the yieldof the full-length product. The O6 modifications may be removed bytreatment with the capping reagent prior to oxidation with I₂/water. Insome instances, inclusion of a capping step during polynucleotidesynthesis decreases the error rate as compared to synthesis withoutcapping. As an example, the capping step comprises treating thesubstrate-bound polynucleotide with a mixture of acetic anhydride and1-methylimidazole. Following a capping step, the device is optionallywashed.

In some instances, following addition of a nucleoside phosphoramidite,and optionally after capping and one or more wash steps, the devicebound growing nucleic acid is oxidized. The oxidation step comprises thephosphite triester is oxidized into a tetracoordinated phosphatetriester, a protected precursor of the naturally occurring phosphatediester internucleoside linkage. In some instances, oxidation of thegrowing polynucleotide is achieved by treatment with iodine and water,optionally in the presence of a weak base (e.g., pyridine, lutidine,collidine). Oxidation may be carried out under anhydrous conditionsusing, e.g. tert-Butyl hydroperoxide or(1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO). In some methods, acapping step is performed following oxidation. A second capping stepallows for device drying, as residual water from oxidation that maypersist can inhibit subsequent coupling. Following oxidation, the deviceand growing polynucleotide is optionally washed. In some instances, thestep of oxidation is substituted with a sulfurization step to obtainpolynucleotide phosphorothioates, wherein any capping steps can beperformed after the sulfurization. Many reagents are capable of theefficient sulfur transfer, including but not limited to3-(Dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-3-thione, DDTT,3H-1,2-benzodithiol-3-one 1,1-dioxide, also known as Beaucage reagent,and N,N,N′N′-Tetraethylthiuram disulfide (TETD).

In order for a subsequent cycle of nucleoside incorporation to occurthrough coupling, the protected 5′ end of the device bound growingpolynucleotide is removed so that the primary hydroxyl group is reactivewith a next nucleoside phosphoramidite. In some instances, theprotecting group is DMT and deblocking occurs with trichloroacetic acidin dichloromethane. Conducting detritylation for an extended time orwith stronger than recommended solutions of acids may lead to increaseddepurination of solid support-bound polynucleotide and thus reduces theyield of the desired full-length product. Methods and compositions ofthe disclosure described herein provide for controlled deblockingconditions limiting undesired depurination reactions. In some instances,the device bound polynucleotide is washed after deblocking. In someinstances, efficient washing after deblocking contributes to synthesizedpolynucleotides having a low error rate.

Methods for the synthesis of polynucleotides typically involve aniterating sequence of the following steps: application of a protectedmonomer to an actively functionalized surface (e.g., locus) to link witheither the activated surface, a linker or with a previously deprotectedmonomer; deprotection of the applied monomer so that it is reactive witha subsequently applied protected monomer; and application of anotherprotected monomer for linking. One or more intermediate steps includeoxidation or sulfurization. In some instances, one or more wash stepsprecede or follow one or all of the steps.

Methods for phosphoramidite-based polynucleotide synthesis comprise aseries of chemical steps. In some instances, one or more steps of asynthesis method involve reagent cycling, where one or more steps of themethod comprise application to the device of a reagent useful for thestep. For example, reagents are cycled by a series of liquid depositionand vacuum drying steps. For substrates comprising three-dimensionalfeatures such as wells, microwells, channels and the like, reagents areoptionally passed through one or more regions of the device via thewells and/or channels.

Methods and systems described herein relate to polynucleotide synthesisdevices for the synthesis of polynucleotides. The synthesis may be inparallel. For example, at least or about at least 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 1000, 10000, 50000, 75000, 100000 or morepolynucleotides can be synthesized in parallel. The total numberpolynucleotides that may be synthesized in parallel may be from2-100000, 3-50000, 4-10000, 5-1000, 6-900, 7-850, 8-800, 9-750, 10-700,11-650, 12-600, 13-550, 14-500, 15-450, 16-400, 17-350, 18-300, 19-250,20-200, 21-150, 22-100, 23-50, 24-45, 25-40, 30-35. Those of skill inthe art appreciate that the total number of polynucleotides synthesizedin parallel may fall within any range bound by any of these values, forexample 25-100. The total number of polynucleotides synthesized inparallel may fall within any range defined by any of the values servingas endpoints of the range. Total molar mass of polynucleotidessynthesized within the device or the molar mass of each of thepolynucleotides may be at least or at least about 10, 20, 30, 40, 50,100, 250, 500, 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,9000, 10000, 25000, 50000, 75000, 100000 picomoles, or more. The lengthof each of the polynucleotides or average length of the polynucleotideswithin the device may be at least or about at least 10, 15, 20, 25, 30,35, 40, 45, 50, 100, 150, 200, 300, 400, 500 nucleotides, or more. Thelength of each of the polynucleotides or average length of thepolynucleotides within the device may be at most or about at most 500,400, 300, 200, 150, 100, 50, 45, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14,13, 12, 11, 10 nucleotides, or less. The length of each of thepolynucleotides or average length of the polynucleotides within thedevice may fall from 10-500, 9-400, 11-300, 12-200, 13-150, 14-100,15-50, 16-45, 17-40, 18-35, 19-25. Those of skill in the art appreciatethat the length of each of the polynucleotides or average length of thepolynucleotides within the device may fall within any range bound by anyof these values, for example 100-300. The length of each of thepolynucleotides or average length of the polynucleotides within thedevice may fall within any range defined by any of the values serving asendpoints of the range.

Methods for polynucleotide synthesis on a surface provided herein allowfor synthesis at a fast rate. As an example, at least 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 125, 150, 175,200 nucleotides per hour, or more are synthesized. Nucleotides includeadenine, guanine, thymine, cytosine, uridine building blocks, oranalogs/modified versions thereof. In some instances, libraries ofpolynucleotides are synthesized in parallel on substrate. For example, adevice comprising about or at least about 100; 1,000; 10,000; 30,000;75,000; 100,000; 1,000,000; 2,000,000; 3,000,000; 4,000,000; or5,000,000 resolved loci is able to support the synthesis of at least thesame number of distinct polynucleotides, wherein polynucleotide encodinga distinct sequence is synthesized on a resolved locus. In someinstances, a library of polynucleotides is synthesized on a device withlow error rates described herein in less than about three months, twomonths, one month, three weeks, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2 days, 24 hours or less. In some instances, larger nucleic acidsassembled from a polynucleotide library synthesized with low error rateusing the substrates and methods described herein are prepared in lessthan about three months, two months, one month, three weeks, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days, 24 hours or less.

In some instances, methods described herein provide for generation of alibrary of nucleic acids comprising variant nucleic acids differing at aplurality of codon sites. In some instances, a nucleic acid may have 1site, 2 sites, 3 sites, 4 sites, 5 sites, 6 sites, 7 sites, 8 sites, 9sites, 10 sites, 11 sites, 12 sites, 13 sites, 14 sites, 15 sites, 16sites, 17 sites 18 sites, 19 sites, 20 sites, 30 sites, 40 sites, 50sites, or more of variant codon sites.

In some instances, the one or more sites of variant codon sites may beadjacent. In some instances, the one or more sites of variant codonsites may not be adjacent and separated by 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more codons.

In some instances, a nucleic acid may comprise multiple sites of variantcodon sites, wherein all the variant codon sites are adjacent to oneanother, forming a stretch of variant codon sites. In some instances, anucleic acid may comprise multiple sites of variant codon sites, whereinnone the variant codon sites are adjacent to one another. In someinstances, a nucleic acid may comprise multiple sites of variant codonsites, wherein some the variant codon sites are adjacent to one another,forming a stretch of variant codon sites, and some of the variant codonsites are not adjacent to one another.

Referring to the Figures, FIG. 3 illustrates an exemplary processworkflow for synthesis of nucleic acids (e.g., genes) from shorternucleic acids. The workflow is divided generally into phases: (1) denovo synthesis of a single stranded nucleic acid library, (2) joiningnucleic acids to form larger fragments, (3) error correction, (4)quality control, and (5) shipment. Prior to de novo synthesis, anintended nucleic acid sequence or group of nucleic acid sequences ispreselected. For example, a group of genes is preselected forgeneration.

Once large nucleic acids for generation are selected, a predeterminedlibrary of nucleic acids is designed for de novo synthesis. Varioussuitable methods are known for generating high density polynucleotidearrays. In the workflow example, a device surface layer is provided. Inthe example, chemistry of the surface is altered in order to improve thepolynucleotide synthesis process. Areas of low surface energy aregenerated to repel liquid while areas of high surface energy aregenerated to attract liquids. The surface itself may be in the form of aplanar surface or contain variations in shape, such as protrusions ormicrowells which increase surface area. In the workflow example, highsurface energy molecules selected serve a dual function of supportingDNA chemistry, as disclosed in International Patent ApplicationPublication WO/2015/021080, which is herein incorporated by reference inits entirety.

In situ preparation of polynucleotide arrays is generated on a solidsupport and utilizes single nucleotide extension process to extendmultiple oligomers in parallel. A deposition device, such as a materialdeposition device, is designed to release reagents in a step wisefashion such that multiple polynucleotides extend, in parallel, oneresidue at a time to generate oligomers with a predetermined nucleicacid sequence 302. In some instances, polynucleotides are cleaved fromthe surface at this stage. Cleavage includes gas cleavage, e.g., withammonia or methylamine.

The generated polynucleotide libraries are placed in a reaction chamber.In this exemplary workflow, the reaction chamber (also referred to as“nanoreactor”) is a silicon coated well, containing PCR reagents andlowered onto the polynucleotide library 303. Prior to or after thesealing 304 of the polynucleotides, a reagent is added to release thepolynucleotides from the substrate. In the exemplary workflow, thepolynucleotides are released subsequent to sealing of the nanoreactor305. Once released, fragments of single stranded polynucleotideshybridize in order to span an entire long range sequence of DNA. Partialhybridization 305 is possible because each synthesized polynucleotide isdesigned to have a small portion overlapping with at least one otherpolynucleotide in the pool.

After hybridization, a PCA reaction is commenced. During the polymerasecycles, the polynucleotides anneal to complementary fragments and gapsare filled in by a polymerase. Each cycle increases the length ofvarious fragments randomly depending on which polynucleotides find eachother. Complementarity amongst the fragments allows for forming acomplete large span of double stranded DNA 306.

After PCA is complete, the nanoreactor is separated from the device 307and positioned for interaction with a device having primers for PCR 308.After sealing, the nanoreactor is subject to PCR 309 and the largernucleic acids are amplified. After PCR 310, the nanochamber is opened311, error correction reagents are added 312, the chamber is sealed 313and an error correction reaction occurs to remove mismatched base pairsand/or strands with poor complementarity from the double stranded PCRamplification products 314. The nanoreactor is opened and separated 315.Error corrected product is next subject to additional processing steps,such as PCR and molecular bar coding, and then packaged 322 for shipment323.

In some instances, quality control measures are taken. After errorcorrection, quality control steps include for example interaction with awafer having sequencing primers for amplification of the error correctedproduct 316, sealing the wafer to a chamber containing error correctedamplification product 317, and performing an additional round ofamplification 318. The nanoreactor is opened 319 and the products arepooled 320 and sequenced 321. After an acceptable quality controldetermination is made, the packaged product 322 is approved for shipment323.

In some instances, a nucleic acid generated by a workflow such as thatin FIG. 3 is subject to mutagenesis using overlapping primers disclosedherein. In some instances, a library of primers are generated by in situpreparation on a solid support and utilize single nucleotide extensionprocess to extend multiple oligomers in parallel. A deposition device,such as a material deposition device, is designed to release reagents ina step wise fashion such that multiple polynucleotides extend, inparallel, one residue at a time to generate oligomers with apredetermined nucleic acid sequence 302.

Computer Systems

Any of the systems described herein, may be operably linked to acomputer and may be automated through a computer either locally orremotely. In various instances, the methods and systems of thedisclosure may further comprise software programs on computer systemsand use thereof. Accordingly, computerized control for thesynchronization of the dispense/vacuum/refill functions such asorchestrating and synchronizing the material deposition device movement,dispense action and vacuum actuation are within the bounds of thedisclosure. The computer systems may be programmed to interface betweenthe user specified base sequence and the position of a materialdeposition device to deliver the correct reagents to specified regionsof the substrate.

The computer system 400 illustrated in FIG. 4 may be understood as alogical apparatus that can read instructions from media 411 and/or anetwork port 405, which can optionally be connected to server 409 havingfixed media 412. The system, such as shown in FIG. 4 can include a CPU401, disk drives 403, optional input devices such as keyboard 415 and/ormouse 416 and optional monitor 407. Data communication can be achievedthrough the indicated communication medium to a server at a local or aremote location. The communication medium can include any means oftransmitting and/or receiving data. For example, the communicationmedium can be a network connection, a wireless connection or an internetconnection. Such a connection can provide for communication over theWorld Wide Web. It is envisioned that data relating to the presentdisclosure can be transmitted over such networks or connections forreception and/or review by a party 422 as illustrated in FIG. 4.

As illustrated in FIG. 5, a high speed cache 504 can be connected to, orincorporated in, the processor 502 to provide a high speed memory forinstructions or data that have been recently, or are frequently, used byprocessor 502. The processor 502 is connected to a north bridge 506 by aprocessor bus 508. The north bridge 506 is connected to random accessmemory (RAM) 510 by a memory bus 512 and manages access to the RAM 510by the processor 502. The north bridge 506 is also connected to a southbridge 514 by a chipset bus 516. The south bridge 514 is, in turn,connected to a peripheral bus 518. The peripheral bus can be, forexample, PCI, PCI-X, PCI Express, or other peripheral bus. The northbridge and south bridge are often referred to as a processor chipset andmanage data transfer between the processor, RAM, and peripheralcomponents on the peripheral bus 518. In some alternative architectures,the functionality of the north bridge can be incorporated into theprocessor instead of using a separate north bridge chip. In someinstances, system 500 can include an accelerator card 522 attached tothe peripheral bus 518. The accelerator can include field programmablegate arrays (FPGAs) or other hardware for accelerating certainprocessing. For example, an accelerator can be used for adaptive datarestructuring or to evaluate algebraic expressions used in extended setprocessing.

Software and data are stored in external storage 524 and can be loadedinto RAM 510 and/or cache 504 for use by the processor. The system 500includes an operating system for managing system resources; non-limitingexamples of operating systems include: Linux, Windows™, MACOS™,BlackBerry OS™, iOS™, and other functionally-equivalent operatingsystems, as well as application software running on top of the operatingsystem for managing data storage and optimization in accordance withexample instances of the present disclosure. In this example, system 500also includes network interface cards (NICs) 520 and 521 connected tothe peripheral bus for providing network interfaces to external storage,such as Network Attached Storage (NAS) and other computer systems thatcan be used for distributed parallel processing.

FIG. 6 is a diagram showing a network 600 with a plurality of computersystems 602 a, and 602 b, a plurality of cell phones and personal dataassistants 602 c, and Network Attached Storage (NAS) 604 a, and 604 b.In example instances, systems 602 a, 602 b, and 602 c can manage datastorage and optimize data access for data stored in Network AttachedStorage (NAS) 604 a and 604 b. A mathematical model can be used for thedata and be evaluated using distributed parallel processing acrosscomputer systems 602 a, and 602 b, and cell phone and personal dataassistant systems 602 c. Computer systems 602 a, and 602 b, and cellphone and personal data assistant systems 602 c can also provideparallel processing for adaptive data restructuring of the data storedin Network Attached Storage (NAS) 604 a and 604 b. FIG. 6 illustrates anexample only, and a wide variety of other computer architectures andsystems can be used in conjunction with the various instances of thepresent disclosure. For example, a blade server can be used to provideparallel processing. Processor blades can be connected through a backplane to provide parallel processing. Storage can also be connected tothe back plane or as Network Attached Storage (NAS) through a separatenetwork interface. In some example instances, processors can maintainseparate memory spaces and transmit data through network interfaces,back plane or other connectors for parallel processing by otherprocessors. In other instances, some or all of the processors can use ashared virtual address memory space.

FIG. 7 is a block diagram of a multiprocessor computer system 700 usinga shared virtual address memory space in accordance with an exampleinstance. The system includes a plurality of processors 702 a-f that canaccess a shared memory subsystem 704. The system incorporates aplurality of programmable hardware memory algorithm processors (MAPs)706 a-f in the memory subsystem 704. Each MAP 706 a-f can comprise amemory 708 a-f and one or more field programmable gate arrays (FPGAs)710 a-f The MAP provides a configurable functional unit and particularalgorithms or portions of algorithms can be provided to the FPGAs 710a-f for processing in close coordination with a respective processor.For example, the MAPs can be used to evaluate algebraic expressionsregarding the data model and to perform adaptive data restructuring inexample instances. In this example, each MAP is globally accessible byall of the processors for these purposes. In one configuration, each MAPcan use Direct Memory Access (DMA) to access an associated memory 708a-f, allowing it to execute tasks independently of, and asynchronouslyfrom the respective microprocessor 702 a-f. In this configuration, a MAPcan feed results directly to another MAP for pipelining and parallelexecution of algorithms.

The above computer architectures and systems are examples only, and awide variety of other computer, cell phone, and personal data assistantarchitectures and systems can be used in connection with exampleinstances, including systems using any combination of generalprocessors, co-processors, FPGAs and other programmable logic devices,system on chips (SOCs), application specific integrated circuits(ASICs), and other processing and logic elements. In some instances, allor part of the computer system can be implemented in software orhardware. Any variety of data storage media can be used in connectionwith example instances, including random access memory, hard drives,flash memory, tape drives, disk arrays, Network Attached Storage (NAS)and other local or distributed data storage devices and systems.

In example instances, the computer system can be implemented usingsoftware modules executing on any of the above or other computerarchitectures and systems. In other instances, the functions of thesystem can be implemented partially or completely in firmware,programmable logic devices such as field programmable gate arrays(FPGAs) as referenced in FIG. 5, system on chips (SOCs), applicationspecific integrated circuits (ASICs), or other processing and logicelements. For example, the Set Processor and Optimizer can beimplemented with hardware acceleration through the use of a hardwareaccelerator card, such as accelerator card 522 illustrated in FIG. 5.

The following examples are set forth to illustrate more clearly theprinciple and practice of embodiments disclosed herein to those skilledin the art and are not to be construed as limiting the scope of anyclaimed embodiments. Unless otherwise stated, all parts and percentagesare on a weight basis.

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the disclosure and are not meant to limit the presentdisclosure in any fashion. The present examples, along with the methodsdescribed herein are presently representative of preferred embodiments,are exemplary, and are not intended as limitations on the scope of thedisclosure. Changes therein and other uses which are encompassed withinthe spirit of the disclosure as defined by the scope of the claims willoccur to those skilled in the art.

Example 1: Functionalization of a Device Surface

A device was functionalized to support the attachment and synthesis of alibrary of polynucleotides. The device surface was first wet cleanedusing a piranha solution comprising 90% H₂SO₄ and 10% H₂O₂ for 20minutes. The device was rinsed in several beakers with DI water, heldunder a DI water gooseneck faucet for 5 min, and dried with N₂. Thedevice was subsequently soaked in NH₄OH (1:100; 3 mL:300 mL) for 5 min,rinsed with DI water using a handgun, soaked in three successive beakerswith DI water for 1 min each, and then rinsed again with DI water usingthe handgun. The device was then plasma cleaned by exposing the devicesurface to O₂. A SAMCO PC-300 instrument was used to plasma etch O₂ at250 watts for 1 min in downstream mode.

The cleaned device surface was actively functionalized with a solutioncomprising N-(3-triethoxysilylpropyl)-4-hydroxybutyramide using aYES-1224P vapor deposition oven system with the following parameters:0.5 to 1 torr, 60 min, 70° C., 135° C. vaporizer. The device surface wasresist coated using a Brewer Science 200× spin coater. SPR™ 3612photoresist was spin coated on the device at 2500 rpm for 40 sec. Thedevice was pre-baked for 30 min at 90° C. on a Brewer hot plate. Thedevice was subjected to photolithography using a Karl Suss MA6 maskaligner instrument. The device was exposed for 2.2 sec and developed for1 min in MSF 26A. Remaining developer was rinsed with the handgun andthe device soaked in water for 5 min. The device was baked for 30 min at100° C. in the oven, followed by visual inspection for lithographydefects using a Nikon L200. A descum process was used to remove residualresist using the SAMCO PC-300 instrument to O₂ plasma etch at 250 wattsfor 1 min.

The device surface was passively functionalized with a 100 μL solutionof perfluorooctyltrichlorosilane mixed with 10 μL light mineral oil. Thedevice was placed in a chamber, pumped for 10 min, and then the valvewas closed to the pump and left to stand for 10 min. The chamber wasvented to air. The device was resist stripped by performing two soaksfor 5 min in 500 mL NMP at 70° C. with ultrasonication at maximum power(9 on Crest system). The device was then soaked for 5 min in 500 mLisopropanol at room temperature with ultrasonication at maximum power.The device was dipped in 300 mL of 200 proof ethanol and blown dry withN₂. The functionalized surface was activated to serve as a support forpolynucleotide synthesis.

Example 2: Synthesis of a 50-Mer Sequence on an OligonucleotideSynthesis Device

A two dimensional oligonucleotide synthesis device was assembled into aflowcell, which was connected to a flowcell (Applied Biosystems (ABI394DNA Synthesizer”). The two-dimensional oligonucleotide synthesis devicewas uniformly functionalized withN-(3-TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE (Gelest) was used tosynthesize an exemplary polynucleotide of 50 bp (“50-merpolynucleotide”) using polynucleotide synthesis methods describedherein.

The sequence of the 50-mer was as described in SEQ ID NO.: 2.5′AGACAATCAACCATTTGGGGTGGACAGCCTTGACCTCTAGACTTCGGCAT ##TTTTTTT TTT3′(SEQ ID NO.: 2), where #denotes Thymidine-succinyl hexamide CEDphosphoramidite (CLP-2244 from ChemGenes), which is a cleavable linkerenabling the release of oligos from the surface during deprotection.

The synthesis was done using standard DNA synthesis chemistry (coupling,capping, oxidation, and deblocking) according to the protocol in Table 3and an ABI synthesizer.

TABLE 3 Synthesis protocols General DNA Synthesis Table 3 Process NameProcess Step Time (sec) WASH (Acetonitrile Wash Acetonitrile SystemFlush 4 Flow) Acetonitrile to Flowcell 23 N2 System Flush 4 AcetonitrileSystem Flush 4 DNA BASE ADDITION Activator Manifold Flush 2(Phosphoramidite + Activator to Flowcell 6 Activator Flow) Activator + 6Phosphoramidite to Flowcell Activator to Flowcell 0.5 Activator + 5Phosphoramidite to Flowcell Activator to Flowcell 0.5 Activator + 5Phosphoramidite to Flowcell Activator to Flowcell 0.5 Activator + 5Phosphoramidite to Flowcell Incubate for 25 sec 25 WASH (AcetonitrileWash Acetonitrile System Flush 4 Flow) Acetonitrile to Flowcell 15 N2System Flush 4 Acetonitrile System Flush 4 DNA BASE ADDITION ActivatorManifold Flush 2 (Phosphoramidite + Activator to Flowcell 5 ActivatorFlow) Activator + 18 Phosphoramidite to Flowcell Incubate for 25 sec 25WASH (Acetonitrile Wash Acetonitrile System Flush 4 Flow) Acetonitrileto Flowcell 15 N2 System Flush 4 Acetonitrile System Flush 4 CAPPING(CapA + B, 1:1, CapA + B to Flowcell 15 Flow) WASH (Acetonitrile WashAcetonitrile System Flush 4 Flow) Acetonitrile to Flowcell 15Acetonitrile System Flush 4 OXIDATION (Oxidizer Oxidizer to Flowcell 18Flow) WASH (Acetonitrile Wash Acetonitrile System Flush 4 Flow) N2System Flush 4 Acetonitrile System Flush 4 Acetonitrile to Flowcell 15Acetonitrile System Flush 4 Acetonitrile to Flowcell 15 N2 System Flush4 Acetonitrile System Flush 4 Acetonitrile to Flowcell 23 N2 SystemFlush 4 Acetonitrile System Flush 4 DEBLOCKING (Deblock Deblock toFlowcell 36 Flow) WASH (Acetonitrile Wash Acetonitrile System Flush 4Flow) N2 System Flush 4 Acetonitrile System Flush 4 Acetonitrile toFlowcell 18 N2 System Flush 4.13 Acetonitrile System Flush 4.13Acetonitrile to Flowcell 15

The phosphoramidite/activator combination was delivered similar to thedelivery of bulk reagents through the flowcell. No drying steps wereperformed as the environment stays “wet” with reagent the entire time.

The flow restrictor was removed from the ABI 394 synthesizer to enablefaster flow. Without flow restrictor, flow rates for amidites (0.1M inACN), Activator, (0.25M Benzoylthiotetrazole (“BTT”; 30-3070-xx fromGlenResearch) in ACN), and Ox (0.02M I2 in 20% pyridine, 10% water, and70% THF) were roughly ˜100 uL/sec, for acetonitrile (“ACN”) and cappingreagents (1:1 mix of CapA and CapB, wherein CapA is acetic anhydride inTHF/Pyridine and CapB is 16% 1-methylimidizole in THF), roughly ˜200uL/sec, and for Deblock (3% dichloroacetic acid in toluene), roughly˜300 uL/sec (compared to ˜50 uL/sec for all reagents with flowrestrictor). The time to completely push out Oxidizer was observed, thetiming for chemical flow times was adjusted accordingly and an extra ACNwash was introduced between different chemicals. After polynucleotidesynthesis, the chip was deprotected in gaseous ammonia overnight at 75psi. Five drops of water were applied to the surface to recoverpolynucleotides. The recovered polynucleotides were then analyzed on aBioAnalyzer small RNA chip.

Example 3: Synthesis of a 100-Mer Sequence on an OligonucleotideSynthesis Device

The same process as described in Example 2 for the synthesis of the50-mer sequence was used for the synthesis of a 100-mer polynucleotide(“100-mer polynucleotide”; 5′CGGGATCCTTATCGTCATCGTCGTACAGATCCCGACCCATTTGCTGTCCACCAGTCATGCTAGCCATACCATGATGATGATGATGATGAGAACCCCGCAT ##TTTTTTTTTT3′, where #denotesThymidine-succinyl hexamide CED phosphoramidite (CLP-2244 fromChemGenes); SEQ ID NO.: 3) on two different silicon chips, the first oneuniformly functionalized withN-(3-TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE and the second onefunctionalized with 5/95 mix of 11-acetoxyundecyltriethoxysilane andn-decyltriethoxysilane, and the polynucleotides extracted from thesurface were analyzed on a BioAnalyzer instrument.

All ten samples from the two chips were further PCR amplified using aforward (5′ATGCGGGGTTCTCATCATC3′; SEQ ID NO.: 4) and a reverse(5′CGGGATCCTTATCGTCATCG3; SEQ ID NO.: 5) primer in a 50 uL PCR mix (25uL NEB Q5 mastermix, 2.5 uL 10 uM Forward primer, 2.5 uL 10 uM Reverseprimer, 1 uL polynucleotide extracted from the surface, and water up to50 uL) using the following thermalcycling program:

98° C., 30 sec

98° C., 10 sec; 63° C., 10 sec; 72° C., 10 sec; repeat 12 cycles

72° C., 2 min

The PCR products were also run on a BioAnalyzer, demonstrating sharppeaks at the 100-mer position. Next, the PCR amplified samples werecloned, and Sanger sequenced. Table 4 summarizes the results from theSanger sequencing for samples taken from spots 1-5 from chip 1 and forsamples taken from spots 6-10 from chip 2.

TABLE 4 Sequencing results Spot Error rate Cycle efficiency 1 1/763 bp99.87% 2 1/824 bp 99.88% 3 1/780 bp 99.87% 4 1/429 bp 99.77% 5 1/1525bp  99.93% 6 1/1615 bp  99.94% 7 1/531 bp 99.81% 8 1/1769 bp  99.94% 91/854 bp 99.88% 10 1/1451 bp  99.93%

Thus, the high quality and uniformity of the synthesized polynucleotideswere repeated on two chips with different surface chemistries. Overall,89% of the 100-mers that were sequenced were perfect sequences with noerrors, corresponding to 233 out of 262.

Table 5 summarizes error characteristics for the sequences obtained fromthe polynucleotide samples from spots 1-10.

TABLE 5 Error characteristics Sample ID/ OSA_ OSA_ OSA_ OSA_ OSA_ OSA_OSA_ OSA_ OSA_ OSA_ Spot no. 0046/1 0047/2 0048/3 0049/4 0050/5 0051/60052/7 0053/8 0054/9 0055/10 Total 32 32 32 32 32 32 32 32 32 32Sequences Sequencing 25 of 27 of 26 of 21 of 25 of 29 of 27 of 29 of 28of 25 of Quality 28 27 30 23 26 30 31 31 29 28 Oligo 23 of 25 of 22 of18 of 24 of 25 of 22 of 28 of 26 of 20 of Quality 25 27 26 21 25 29 2729 28 25 ROI 2500 2698 2561 2122 2499 2666 2625 2899 2798 2348 MatchCount ROI 2 2 1 3 1 0 2 1 2 1 Mutation ROI Multi 0 0 0 0 0 0 0 0 0 0Base Deletion ROI Small 1 0 0 0 0 0 0 0 0 0 Insertion ROI 0 0 0 0 0 0 00 0 0 Single Base Deletion Large 0 0 1 0 0 1 1 0 0 0 Deletion CountMutation: 2 2 1 2 1 0 2 1 2 1 G > A Mutation: 0 0 0 1 0 0 0 0 0 0 T > CROI Error 3 2 2 3 1 1 3 1 2 1 Count ROI Error Err: ~1 Err: ~1 Err: ~1Err: ~1 Err: ~1 Err: ~1 Err: ~1 Err: ~1 Err: ~1 Err: ~1 Rate in 834 in1350 in 1282 in 708 in 2500 in 2667 in 876 in 2900 in 1400 in 2349 ROIMP Err: MP Err: MP Err: MP Err: MP Err: MP Err: MP Err: MP Err: MP Err:MP Err: Minus ~1 in ~1 in ~1 in ~1 in ~1 in ~1 in ~1 in ~1 in ~1 in ~1in Primer 763 824 780 429 1525 1615 531 1769 854 1451 Error Rate

Example 4: Design of Antibody Scaffolds

To generate scaffolds, structural analysis, repertoire sequencinganalysis of the heavy chain, and specific analysis of heterodimerhigh-throughput sequencing datasets were performed. Each heavy chain wasassociated with each light chain scaffold. Each heavy chain scaffold wasassigned 5 different long CDRH3 loop options. Each light chain scaffoldwas assigned 5 different L3 scaffolds. The heavy chain CDRH3 stems werechosen from the frequently observed long H3 loop stems (10 amino acidson the N-terminus and the C-terminus) found both across individuals andacross V-gene segments. The light chain scaffold L3s were chosen fromheterodimers comprising long H3s. Direct heterodimers based oninformation from the Protein Data Bank (PDB) and deep sequencingdatasets were used in which CDR H1, H2, L1, L2, L3, and CDRH3 stems werefixed. The various scaffolds were then formatted for display on phage toassess for expression.

Structural Analysis

About 2,017 antibody structures were analyzed from which 22 structureswith long CDRH3s of at least 25 amino acids in length were observed. Theheavy chains included the following: IGHV1-69, IGHV3-30, IGHV4-49, andIGHV3-21. The light chains identified included the following: IGLV3-21,IGKV3-11, IGKV2-28, IGKV1-5, IGLV1-51, IGLV1-44, and IGKV1-13. In theanalysis, four heterodimer combinations were observed multiple timesincluding: IGHV4-59/61-IGLV3-21, IGHV3-21-IGKV2-28, IGHV1-69-IGKV3-11,and IGHV1-69-IGKV1-5. An analysis of sequences and structures identifiedintra-CDRH3 disulfide bonds in a few structures with packing of bulkyside chains such as tyrosine in the stem providing support for long H3stability. Secondary structures including beta-turn-beta sheets and a“hammerhead” subdomain were also observed.

Repertoire Analysis

A repertoire analysis was performed on 1,083,875 IgM+/CD27-naïve B cellreceptor (BCR) sequences and 1,433,011 CD27+ sequences obtained byunbiased 5′RACE from 12 healthy controls. The 12 healthy controlscomprised equal numbers of male and female and were made up of 4Caucasian, 4 Asian, and 4 Hispanic individuals. The repertoire analysisdemonstrated that less than 1% of the human repertoire comprises BCRswith CDRH3s longer than 21 amino acids. A V-gene bias was observed inthe long CDR3 subrepertoire, with IGHV1-69, IGHV4-34, IGHV1-18, andIGHV1-8 showing preferential enrichment in BCRs with long H3 loops. Abias against long loops was observed for IGHV3-23, IGHV4-59/61,IGHV5-51, IGHV3-48, IGHV3-53/66, IGHV3-15, IGHV3-74, IGHV3-73, IGHV3-72,and IGHV2-70. The IGHV4-34 scaffold was demonstrated to be autoreactiveand had a short half-life.

Viable N-terminal and C-terminal CDRH3 scaffold variation for long loopswere also designed based on the 5′RACE reference repertoire. About81,065 CDRH3s of amino acid length 22 amino acids or greater wereobserved. By comparing across V-gene scaffolds, scaffold-specific H3stem variation was avoided as to allow the scaffold diversity to becloned into multiple scaffold references.

Heterodimer Analysis

Heterodimer analysis was performed on scaffolds. Variant sequences andlengths of the scaffolds were assayed.

Structural Analysis

Structural analysis was performed using GPCR scaffolds of variantsequences and lengths were assayed.

Example 5: Generation of GPCR Antibody Libraries

Based on GPCR-ligand interaction surfaces and scaffold arrangements,libraries were designed and de novo synthesized. See Example 4. 10variant sequences were designed for the variable domain, heavy chain,237 variant sequences were designed for the heavy chain complementaritydetermining region 3, and 44 variant sequences were designed for thevariable domain, light chain. The fragments were synthesized as threefragments following similar methods as described in Examples 1-3.

Following de novo synthesis, 10 variant sequences were generated for thevariable domain, heavy chain, 236 variant sequences were generated forthe heavy chain complementarity determining region 3, and 43 variantsequences were designed for a region comprising the variable domain,light chain and CDRL3 and of which 9 variants for variable domain, lightchain were designed. This resulted in a library with about 10⁵ diversity(10×236×43). This was confirmed using next generation sequencing (NGS)with 16 million reads.

The various light and heavy chains were then tested for expression andprotein folding. The 10 variant sequences for variable domain, heavychain included the following: IGHV1-18, IGHV1-69, IGHV1-8 IGHV3-21,IGHV3-23, IGHV3-30/33rn, IGHV3-28, IGHV3-74, IGHV4-39, and IGHV4-59/61.Of the 10 variant sequences, IGHV1-18, IGHV1-69, and IGHV3-30/33rnexhibited improved characteristics such as improved thermostability. 9variant sequences for variable domain, light chain included thefollowing: IGKV1-39, IGKV1-9, IGKV2-28, IGKV3-11, IGKV3-15, IGKV3-20,IGKV4-1, IGLV1-51, and IGLV2-14. Of the 9 variant sequences, IGKV1-39,IGKV3-15, IGLV1-51, and IGLV2-14 exhibited improved characteristics suchas improved thermostability.

Example 6: GPCR Libraries

This example describes the generation of GPCR libraries.

Materials and Method

Stable Cell Line and Phage Library Generation

The full length human GLP-1R gene (UniProt—P43220) with an N-terminalFLAG tag and C-terminal GFP tag cloned into pCDNA3.1(+) vector(ThermoFisher) was transfected into suspension Chinese Hamster Ovary(CHO) cells to generate the stable cell line expressing GLP-1R. Targetexpression was confirmed by FACS. Cells expressing >80% of GLP-1R by GFPwere then directly used for cell-based selections.

Germline heavy chain IGHV1-69, IGHV3-30 and germline light chainIGKV1-39, IGKV3-15, IGLV1-51, IGLV2-14 framework combinations were usedin the GPCR-focused phage-displayed library, and all six CDR diversitieswere encoded by oligo pools synthesized similar to Examples 1-3 above.The CDRs were also screened to ensure they did not containmanufacturability liabilities, cryptic splice sites, or commonly usednucleotide restriction sites. The heavy chain variable region (VH) andlight chain variable region (VL) were linked by (G4S)3 linker (SEQ IDNO: 718). The resulting scFv (VH-linker-VL) gene library was cloned intoa pADL 22-2c (Antibody Design Labs) phage display vector by NotIrestriction digestion and electroporated into TG1 electro-competent E.coli cells. (Lucigen). The final library has a diversity of 1.1×10¹⁰size which was verified by NGS.

Panning and Screening Strategy Used to Isolate Agonist GLP-1R scFvClones

Before panning on GLP-1R expressing CHO cells, phage particles wereblocked with 5% BSA/PBS and depleted for non-specific binders on CHOparent cells. For CHO parent cell depletion, the input phage aliquot wasrotated at 14 rpm/min with 1×10⁸ CHO parent cells for 1 hour at roomtemperature (RT). The cells were then pelleted by centrifuging at 1,200rpm for 10 mins in a tabletop Eppendorf centrifuge 5920RS/4×1000 rotorto deplete the non-specific CHO cell binders. The phage supernatant,depleted of CHO cell binders, was then transferred to 1×10⁸ GLP-1Rexpressing CHO cells. The phage supernatant and GLP-1R expressing CHOcells were rotated at 14 rpm/min for 1 hour at RT to select for GLP-1Rbinders. After incubation, the cells were washed several times with1×PBS/0.5% Tween to remove non-binding clones. To elute the phage boundto the GLP-1R cells, the cells were incubated with trypsin in PBS bufferfor 30 minutes at 37° C. The cells were pelleted by centrifuging at1,200 rpm for 10 mins. The output supernatant enriched in GLP-1R bindingclones was amplified in TG1 E. coli cells to use as input phage for thenext round of selection. This selection strategy was repeated for fiverounds. Every round was depleted against the CHO parent background.Amplified output phage from a round was used as the input phage for thesubsequent round, and the stringency of washes were increased in eachsubsequent round of selections with more washes. After five rounds ofselection, 500 clones from each of round 4 and round 5 were Sangersequenced to identify unique clones.

Next-Generation Sequencing Analysis

The phagemid DNA was miniprepped from the output bacterial stocks of allpanning rounds. The variable heavy chain (VH) was PCR amplified from thephagemid DNA using the Forward Primer ACAGAATTCATTAAAGAGGAGAAATTAACC(SEQ ID NO: 719) and reverse primer TGAACCGCCTCCACCGCTAG (SEQ ID NO:720). The PCR product was directly used for library preparation usingthe KAPA HyperPlus Library Preparation Kit (Kapa Biosystems, product#KK8514). To add diversity in the library, the samples were spiked with15% PhiX Control purchased from Illumina, Inc. (product #FC-110-3001).The library was then loaded onto Illumina's 600 cycle MiSeq Reagent Kitv3 (Illumina, product #MS-102-3003) and run on the MiSeq instrument.

Reformatting and High Throughput (HT) IgG Purification

Expi293 cells were transfected using Expifectamine (ThermoFisher,A14524) with the heavy chain and light chain DNA at a 2:1 ratio andsupernatants were harvested 4 days post-transfection before cellviability dropped below 80%. Purifications were undertaken using eitherKing Fisher (ThermoFisher) with protein A magnetic beads or Phynexusprotein A column tips (Hamilton). For large scale production of IgGclones that were evaluated in in vivo mouse studies an Akta HPLCpurification system (GE) was used.

IgG characterization and quality control. The purified IgGs for thepositive GLP-1R binders (hits) were subjected to characterization fortheir purity by LabChip GXII Touch HT Protein Express high-sensitivityassay. Dithiothreitol (DTT) was used to reduce the IgG into VH and VL.IgG concentrations were measured using Lunatic (UnChain). IgG for invivo mouse studies were further characterized by HPLC and tested forendotoxin levels (Endosafe® nexgen-PTS™ Endotoxin Testing, CharlesRiver), with less than 5 EU per kg dosing.

Binding Assays and Flow Cytometry

GLP-1R IgG clones were tested in a binding assay coupled to flowcytometry analysis as follows: FLAG-GLP-1R-GFP expressing CHO cells(CHO-GLP-1R) and CHO-parent cells were incubated with 100 nM IgG for 1 hon ice, washed three times and incubated with Alexa 647 conjugatedgoat-anti-human antibody (1:200) (Jackson ImmunoResearch Laboratories,109-605-044) for 30 min on ice, followed by three washes, centrifugingto pellet the cells between each washing step. All incubations andwashes were in buffer containing PBS+1% BSA. For titrations, IgG wasserially diluted 1:3 starting from 100 nM down to 0.046 nM. Cells wereanalyzed by flow cytometry and hits (a hit is an IgG that specificallybinds to CHO-GLP-1R) were identified by measuring the GFP signal againstthe Alexa 647 signal. Flow cytometry data of binding assays with 100 nMIgG are presented as dot plots. Analyses of binding assays with IgGtitrations are presented as binding curves plotting IgG concentrationsagainst MFI (mean fluorescence intensity).

Ligand Competition Assay

Ligand Competition Assays Involved Co-Incubating the Primary IgG with 1μM GLP-1 (7-36). For Each Data Point, IgG (600 nM) was Prepared in FlowBuffer (PBS+1% BSA) and Diluted 1:3 Down for 8 Titration Points. PeptideGLP-1 7-36 (2 μM) was Prepared Similarly with the Flow Buffer (PBS+1%BSA). Each Well Contained 100,000 Cells to which 50 μL of IgG and 504 ofPeptide (=Plus) or Buffer Alone without Peptide (=Minus) were Added.Cells and IgG/Peptide Mix were Incubated for Lhr on Ice, and afterWashing, Secondary Antibody (Goat Anti-Human APC, Jackson ImmunoResearchLaboratories, Product #109-605-044) Diluted 1:200 in PBS+1% BSA wasAdded. This was Incubated on Ice for 30 Mins (504 Per Well), BeforeWashing and Resuspending in 604 Buffer. Finally, the Assay Read-Out wasMeasured on an Intellicyt® IQue3 Screener at a Rate of 4 Seconds PerWell.

Results

Design of GPCR Focused Antibody Library is Based on GPCR Binding Motifsand GPCR Antibodies

All known GPCR interactions, which include interactions of GPCRs withligands, peptides, antibodies, endogenous extracellular loops and smallmolecules were analyzed to map the GPCR binding molecular determinants.Crystal structures of almost 150 peptides, ligand or antibodies bound toECDs of around 50 GPCRs (http://www.gperdb.org) were used to identifyGPCR binding motifs. Over 1000 GPCR binding motifs were extracted fromthis analysis. In addition, by analysis of all solved structures ofGPCRs (zhanglab.ccmb.med.umich.edu/GPCR-EXP/), over 2000 binding motifsfrom endogenous extracellular loops of GPCRs were identified. Finally,by analysis of structures of over 100 small molecule ligands bound toGPCR, a reduced amino acid library of 5 amino acids (Tyr, Phe, His, Proand Gly) that may be able to recapitulate many of the structuralcontacts of these ligands was identified. A sub-library with thisreduced amino acid diversity was placed within a CxxxxxC motif. Intotal, over 5000 GPCR binding motifs were identified (FIGS. 9A-9E).These binding motifs were placed in one of five different stem regions:CARDLRELECEEWTxxxxxSRGPCVDPRGVAGSFDVW (SEQ ID NO: 721),CARDMYYDFxxxxxEVVPADDAFDIW (SEQ ID NO: 722),CARDGRGSLPRPKGGPxxxxxYDSSEDSGGAFDIW (SEQ ID NO: 723),CARANQHFxxxxxGYHYYGMDVW (SEQ ID NO: 724), CAKHMSMQxxxxxRADLVGDAFDVW (SEQID NO: 725).

These stem regions were selected from structural antibodies withultra-long HCDR3s. Antibody germlines were specifically chosen totolerate these ultra-long HCDR3s. Structure and sequence analysis ofhuman antibodies with longer than 21 amino acids revealed a V-gene biasin antibodies with long CDR3s. Finally, the germline IGHV (IGHV1-69 andIGHV3-30), IGKV (IGKV1-39 and IGKV3-15) and IGLV (IGLV1-51 and IGLV2-14)genes were chosen based on this analysis.

In addition to HCDR3 diversity, limited diversity was also introduced inthe other 5 CDRs. There were 416 HCDR1 and 258 HCDR2 variants in theIGHV1-69 domain; 535 HCDR1 and 416 HCDR2 variants in the IGHV3-30domain; 490 LCDR1, 420 LCDR2 and 824 LCDR3 variants in the IGKV1-39domain; 490 LCDR1, 265 LCDR2 and 907 LCDR3 variants in the IGKV3-15domain; 184 LCDR1, 151 LCDR2 and 824 LCDR3 variants in the IGLV1-51domain; 967 LCDR1, 535 LCDR2 and 922 LCDR3 variants in the IGLV2-14domain (FIG. 10). These CDR variants were selected by comparing thegermline CDRs with the near-germline space of single, double and triplemutations observed in the CDRs within the V-gene repertoire of at leasttwo out of 12 human donors. All CDRs have were pre-screened to removemanufacturability liabilities, cryptic splice sites or nucleotiderestriction sites. The CDRs were synthesized as an oligo pool andincorporated into the selected antibody scaffolds. The heavy chain (VH)and light chain (VL) genes were linked by (G₄S)₃ linker (SEQ ID NO:718). The resulting scFv (VH-linker-VL) gene pool was cloned into aphagemid display vector at the N-terminal of the M13 gene-3 minor coatprotein. The final size of the GPCR library is 1×10¹⁰ in a scFv format.Next-generation sequencing (NGS) was performed on the final phagelibrary to analyze the HCDR3 length distribution in the library forcomparison with the HCDR3 length distribution in B-cell populations fromthree healthy adult donors. The HCDR3 sequences from the three healthydonors used were derived from a publicly available database with over 37million B-cell receptor sequences³¹. The HCDR3 length in the GPCRlibrary is much longer than the HCDR3 length observed in B-cellrepertoire sequences. On average, the median HCDR3 length in the GPCRlibrary (which shows a biphasic pattern of distribution) is two or threetimes longer (33 to 44 amino acids) than the median lengths observed innatural B-cell repertoire sequences (15 to 17 amino acids) (FIG. 11).The biphasic length distribution of HCDR3 in the GPCR library is mainlycaused by the two groups of stems (8aa, 9aaxxxxx10aa, 12aa) and (14aa,16aa xxxxx18aa, 14aa) used to present the motifs within HCDR3.

Example 7: VHH Libraries

Synthetic VHH libraries were developed. For the ‘VHH Ratio’ library withtailored CDR diversity, 2391 VHH sequences (iCAN database) were alignedusing Clustal Omega to determine the consensus at each position and theframework was derived from the consensus at each position. The CDRs ofall of the 2391 sequences were analyzed for position-specific variation,and this diversity was introduced in the library design. For the ‘VHHShuffle’ library with shuffled CDR diversity, the iCAN database wasscanned for unique CDRs in the nanobody sequences. 1239 unique CDR1's,1600 unique CDR2's, and 1608 unique CDR3's were identified and theframework was derived from the consensus at each framework positionamongst the 2391 sequences in the iCAN database. Each of the uniqueCDR's was individually synthesized and shuffled in the consensusframework to generate a library with theoretical diversity of 3.2×10⁹.The library was then cloned in the phagemid vector using restrictionenzyme digest. For the ‘VHH hShuffle’ library (a synthetic “human” VHHlibrary with shuffled CDR diversity), the iCAN database was scanned forunique CDRs in the nanobody sequences. 1239 unique CDR1's, 1600 uniqueCDR2's, and 1608 unique CDR3's were identified and framework 1, 3, and 4was derived from the human germline DP-47 framework. Framework 2 wasderived from the consensus at each framework position amongst the 2391sequences in the iCAN database. Each of the unique CDR's wasindividually synthesized and shuffled in the partially humanizedframework using the NUGE tool to generate a library with theoreticaldiversity of 3.2×10⁹. The library was then cloned in the phagemid vectorusing the NUGE tool.

The Carterra SPR system was used to assess binding affinity and affinitydistribution for VHH-Fc variants. VHH-Fc demonstrate a range ofaffinities for TIGIT, with a low end of 12 nM K_(D) and a high end of1685 nM K_(D) (data not shown). FIG. 12 provides specific values for theVHH-Fc clones for ELISA, Protein A (mg/ml), and K_(D) (nM).

Example 8. Hyperimmune Immunoglobulin Library for A2A Receptor

A hyperimmune immunoglobulin (IgG) library was created using similarmethods as described in Example 7. Briefly, the hyperimmune IgG librarywas generated from analysis of databases of human naïve and memoryB-cell receptor sequences consisting of more than 37 million unique IgHsequences from each of 3 healthy donors. More than two million CDRH3sequences were gathered from the analysis and individually constructedusing methods similar to Examples 1-3. The CDRH3 sequences wereincorporated into the VHH hShuffle library described in Example 9. Thefinal library diversity was determined to be 1.3×10¹⁰. A schematic ofthe design can be seen in FIG. 13.

73 out of 88 unique clones had a target cell MFI values 2 fold overparental cells. 15 out of 88 unique Clones with target cell MFI values20 fold over parental cells. Data for adenosine A2A receptor variantA2AR-90-007 is seen in FIGS. 14A-14B.

This Example shows generation of a VHH library for the A2AR with highaffinity and K_(D) values in the sub-nanomolar range.

Example 9. GPCR Libraries with Varied CDR's

A GPCR library was created using a CDR randomization scheme.

Briefly, GPCR libraries were designed based on GPCR antibody sequences.Over sixty different GPCR antibodies were analyzed and sequences fromthese GPCRs were modified using a CDR randomization scheme.

The heavy chain IGHV3-23 design is seen in FIG. 15A. As seen in FIG.15A, IGHV3-23 CDRH3's had four distinctive lengths: 23 amino acids, 21amino acids, 17 amino acids, and 12 amino acids, with each length havingits residue diversity. The ratio for the four lengths were thefollowing: 40% for the CDRH3 23 amino acids in length, 30% for the CDRH321 amino acids in length, 20% for the CDRH3 17 amino acids in length,and 10% for the CDRH3 12 amino acids in length. The CDRH3 diversity wasdetermined to be 9.3×10⁸, and the full heavy chain IGHV3-23 diversitywas 1.9×10¹³.

The heavy chain IGHV1-69 design is seen in FIG. 15B. As seen in FIG.15B, IGHV1-69 CDRH3's had four distinctive lengths: 20 amino acids, 16amino acids, 15 amino acids, and 12 amino acids, with each length havingits residue diversity. The ratio for the four lengths were thefollowing: 40% for the CDRH3 20 amino acids in length, 30% for the CDRH316 amino acids in length, 20% for the CDRH3 15 amino acids in length,and 10% for the CDRH3 12 amino acids in length. The CDRH3 diversity wasdetermined to be 9×10⁷, and the full heavy chain IGHV-69 diversity is4.1×10¹².

The light chains IGKV 2-28 and IGLV 1-51 design is seen in FIG. 15C.Antibody light chain CDR sequences were analyzed for position-specificvariation. Two light chain frameworks were selected with fixed CDRlengths. The theoretical diversities were determined to be 13800 and5180 for kappa and light chains, respectively.

The final theoretical diversity was determined to be 4.7×10¹⁷ and thefinal, generated Fab library had a diversity of 6×10⁹. See FIG. 15D.

Example 10. Adenosine A2A Receptor Libraries with Varied CDR's

An adenosine A2A receptor library is created using a CDR randomizationscheme similarly described in Example 9.

Briefly, adenosine A2A receptor libraries are designed based on GPCRantibody sequences. Over sixty different GPCR antibodies are analyzedand sequences from these GPCRs are modified using a CDR randomizationscheme. Adenosine A2A receptor variant IgGs designed using the CDRrandomization scheme are purified and are assayed to determinecell-based affinity measurements and for functional analysis.

Example 11. A2A Variant Immunoglobulins

A2AR variant immunoglobulins generated were assayed in variousfunctional assays.

First, A2AR immunoglobulin scFv phage libraries were panned on cells andimmobilized A2a proteins, and screened. The output phage numbers fromeach round of selection are seen in Tables 7-8.

TABLE 7 Target Library Round 1 Round 2 Round 3 Round 4 Round 5 HEK293-Mouse 2.7 × 10⁶ 4.1 × 10⁷ 5.0 × 10⁷ 5.0 × 10⁷ 1.2 × 10⁸ A2a Cells ImmuneA2a protein Humanized 4.1 × 10⁶ 8.0 × 10⁷ 2.3 × 10⁸ 1.2 × 10⁷ 5.8 × 10⁷Synthetic A2a protein + Humanized 5.2 × 10⁶ 4.5 × 10⁷ 1.3 × 10⁸ 3.0 ×10⁷ 6.7 × 10⁷ ZM241385 Synthetic A2a protein Mouse 4.3 × 10⁷ 5.8 × 10⁷3.0 × 10⁷ 4.8 × 10⁷ 3.2 × 10⁷ Immune A2a protein + Mouse 2.4 × 10⁷ 3.7 ×10⁷ 1.9 × 10⁸ 6.0 × 10⁷ 6.0 × 10⁶ ZM241385 Immune

TABLE 8 Target Library Round 1 Round 2 Round 3 Round 4 Round 5 HEK293-Immune 1.3 × 10⁶ 3.1 × 10⁷ 5.0 × 10⁷ 5.0 × 10⁷ 1.3 × 10⁸ A2a Cells A2aprotein Synthetic 5.2 × 10⁶ 3.7 × 10⁷ 1.5 × 10⁸ 1.2 × 10⁷ 4.9 × 10⁷ A2aprotein + Synthetic 6.7 × 10⁶ 2.9 × 10⁷ 6.0 × 10⁷ 2.0 × 10⁷ 6.0 × 10⁷ZM241385 A2a protein Immune 8.0 × 10⁶ 2.0 × 10⁷ 9.0 × 10⁷ 2.3 × 10⁷ 2.8× 10⁷ A2a protein + Immune 6.0 × 10⁶ 1.7 × 10⁷ 1.3 × 10⁸ 4.6 × 10⁷ 1.9 ×10⁷ ZM241385

Example 12. Screening Antibody Binding

Selected A2AR immunoglobulins from the groups listed in Tables 15-18were assayed for binding to the targets as listed in the tables.

HEK293-A2a Cells

Flow cytometry data showing binding to HEK293-A2a cells usingimmunoglobulins from variant libraries were generated using 100 nM IgGand compared to detected binding in parent cells. Binding using variantsfrom an immune library are shown in FIGS. 16A-16N. A control is shown inFIG. 16O, showing cell binding with Human Adenosine A2aR monoclonal(MAB9497). Selected variants were assessed for binding at concentrationstitrated from 100 nM. Resulting curves are show in FIGS. 17A-17H.Binding curves are plotted with IgG concentration vs. MFI (meanfluorescence intensity). Binding using variants from a mouse immunelibrary are shown in FIGS. 18A-18N. A control is shown in FIG. 18O,showing cell binding with Human Adenosine A2aR monoclonal (MAB9497).Selected variants were assessed for binding at concentrations titratedfrom 100 nM. Resulting curves are show in FIGS. 19A-19G. Binding curvesare plotted with IgG concentration vs. MFI (mean fluorescenceintensity).

Protein Bindings

Purified A2a immunoglobulins from Tables 15-18 were assayed for bindingin a titration from 100 nM. Results of selected variants are shown inFIGS. 20A-20G.

Example 13. Agonist Response in LANCE® cAMP Assay

An agonist dose-response assay was performed using a LANCE® cAMP assayin 384-well format using 2500 cells/well according to manufacturer'sinstructions. Cell stimulation with NECA and CGS 21680 was performed for30 min at room temperature. Readings were taken on a EnVision platereader in Laser mode. Data is shown in FIG. 21. The Z′-factor wascalculated for NECA with at least 16 background and 16 maximal signalpoints (Z′=0.80). Calculated EC₅₀ (M) for NECA=2.7×10⁻⁷ and for CGS21680=4.3×10⁻⁷.

Example 14. Antagonist Response in LANCE® cAMP Assay

An antagonist dose-response assay was performed using a LANCE® cAMPassay in 384-well format using 2500 cells/well and 1 μM NECA (referenceagonist) according to manufacturer's instruction. Cell stimulation withZM241385 was performed for 30 min at room temperature. Readings weretaken on a EnVision plate reader in Laser mode. Data is shown in FIG.22. Calculated IC₅₀ (M) for ZM241385=1.25×10⁻⁵.

Example 15. A2A cAMP Antagonist Titration

Cells were plated at 3000/well and pre-incubated with fixed 100 nM IgGfor 1 hr at room temperature, followed by stimulation with NECAtitration for 30 min at room temperature according to manufacturer'sinstructions. Buffer was PBS+0.1% BSA+0.5 mM IBMX. Results shown in FIG.23. Absolute IC50 is shown in Table 9, indicating A2A-1 is a negativeallosteric modulator.

TABLE 9 +no Ab A2A − 1 R&D control antibody IC50 0.03040 0.2816 2.253

Example 16. LANCE® Allosteric cAMP Assay

A2A-1 and A2A-9 were assayed for allosteric modulation. Cells werepre-incubated with titrated IgG for 1 hr at room temperature, followedby stimulation with fixed NECA concentration. Results are shown in FIG.24. IC50 values are shown in Table 10, indicating A2A-1 is a negativeallosteric modulator.

TABLE 10 A2A − 1 A2A − 9 R&D control antibody Absolute IC50 1.833 4.1069.432

Example 17. cAMP Allosteric A2A Perkin Elmer

A2A-9 was assayed as described in Example 15. Resulting response curvesare shown in FIG. 25. Calculated IC50 for A2A-9 is Shown in Table 11.

TABLE 11 A2A − 9 R&D control antibody No antibody Absolute IC50 ~0.4513~0.5126 ~0.2556

Example 18. A2A cAMP Antagonist Titration

A2A-9 was assayed as described in Example 16. Resulting response curvesare shown in FIG. 26. Calculated IC50 values are shown in Table 12.Results indicate A2A-9 is an antagonist.

TABLE 12 A2A − 9 R&D control antibody Absolute IC50 4.106 9.432

Example 19. A2A Antagonistic cAMP Assay

Selected variants were assayed for binding to target. Immunoglobulinswere titrated in triplicate and incubated on cells for 1 hour, followedby incubation with 0.5 μM NECA for 30 minutes. Binding curves showingrelative fluorescent units (RFU) ratio at 665 nm/615 nm versus nM IgG ona log scale are shown in FIGS. 27A-27C. Final binding studies foundfunctional antibodies in the generated libraries as listed in Table 13and Table 14.

TABLE 13 Target Library Reformatted Functional HEK293-A2a Cells MouseImmune 14 A2a protein Humanized Synthetic 95 2 A2a protein + ZM241385Humanized Synthetic 95 3 A2a protein Mouse Immune 12 1 A2a protein +ZM241385 Mouse Immune 22 0

TABLE 14 Target Library Reformatted Functional HEK293-A2a Cells Immune14 A2a protein Synthetic 95 2 A2a protein + ZM241385 Synthetic 95 5 A2aprotein Immune 29 4 A2a protein + ZM241385 Immune 10 5

Example 20. A2AR Cell Functional cAMP Assays

Allosteric and Antagonistic cAMP Assays were Performed Using A2A CellLines

Briefly, cells were pre-incubated with anti-A2AR antibody at 100 nMfollowed by NECA stimulation 3× titration from 100 uM. Data from afunctional allosteric cAMP assay is seen in FIGS. 28A-28C. ZM241385functioned as an antagonist. “No Ab” functioned as agonist only.

For a functional antagonistic cAMP assays, cells were pre-incubated withanti-A2AR antibody 3× titration from 100 nM followed by NECA stimulationat 0.5 uM. Data is seen in FIGS. 29A-29C. Cells were also pre-incubatedwith anti-A2AR antibody 3× titration from 100 nM followed by NECAstimulation at 10 uM. Data is seen in FIGS. 30A-30C.

Based on the data, for NECA titration, IgG titration (NECA 0.5 uM), andIgG titration (NECA 10 uM), A2AR variant A2A-17, A2A-19, A2A-24, A2A-26,and A2A-27 exhibited improved function in cAMP assays.

Example 21. T Cell Activation Assays

Variant A2A-77 developed according to the previous examples was found tobe a high affinity binder for hA2a (FIG. 31A). A2A-77 was determined tobe a functional antagonist in vitro (FIG. 31B) and had high specificityin vitro (FIG. 31C). A2A-77 was found to bind to cynomolgus PBMC withcells expressing A2AR, including T cell, NK cell, dendritic cell, andmacrophage (FIG. 31D). Further studies were performed to determineeffects in T-cell activation assays.

Briefly, 2×10⁵ PBMCs per well were incubated with antagonist ZM-241385or A2aR immunoglobulins that were titrated from 100 nM for 30 minutes at37° C., followed by treatment with A2AR agonist NECA at 1 μM for 30minutes at 37° C. The cells were then activated by magnetic beadscoating with anti-CD3ε/CD28 antibodies. Three days after incubation, thesupernatant was collected for detecting IFN-γ release and evaluating Tcell activation. ZM-241385 is potent and used as a selective smallmolecule A2A antagonist control.

Data is seen in FIGS. 32A-32H. As seen in FIGS. 32A-32B, T cellactivation was observed with variants A2A-81, A2A-51, A2A-53, A2A-77,A2A-31, and A2A-78. A2A-77 was further observed to have an IC₅₀ of 5.92nM (FIG. 32C). Data in FIGS. 32D-32G demonstrate while using more NECAto suppress T cell activation, A2A-77 and A2A-81 cannot restore T cellactivation as in low NECA. A2A-51 still works well in high NECA.

This Example shows that A2A-77 is a functional antagonist of A2AR toblock immunosuppression.

Example 22. Exemplary Sequences

TABLE 15  Variable Heavy Chain CDRs SEQ SEQ SEQ A2AR ID CDR1 ID CDR2 IDCDR3 Variant NO Sequence NO Sequence NO Sequence A2A-1  6 GGSISSSN 95YPSGN 184 DEGY A2A-2  7 GYTFTGY 96 NPNSGG 185 GGPFDY A2A-3  8 GYTFTGY 97NPNSGG 186 VYSYGFDY A2A-4  9 GFTFSDY 98 SSSGST 187 DNWAFDL A2A-5 10GFTFSSY 99 SSSSSY 188 TWYSSSPFDY A2A-6 11 GFTFSNY 100 SSSSSY 189DSGSYYDWFDP A2A-7 12 GFTFSSY 101 SGSGGS 190 YSNYFDY A2A-8 13 GYSITSGY102 SYDGS 191 VHHYYGSSYFDY A2A-9 14 GYSITSGY 103 RYDGS 192 VHHYYGSSYFDYA2A-10 15 GYSITSGY 104 SYDGS 193 DPYYYGSSYFDY A2A-11 16 GFTFSDY 105NYDGSS 194 EYYYGSSSFAY A2A-12 17 GFTFNDY 106 NYDGSS 195 EYYYGSSSFAYA2A-13 18 GFTFSDF 107 SSGSST 196 REFAY A2A-14 19 GFTFSDY 108 SSGSGT 197PNYHGSSPFAY A2A-15 20 GFTFSTY 109 SGSGGS 198 ARGKWRWRLGRRYDY A2A-16 21GFTFNNY 110 SGSGGD 199 ARGYWRWRLLRRYDY A2A-17 22 GFNIGNT 111 NPNYGT 200DYGSSSFDY A2A-18 23 GFSFSGY 112 SGSGGS 201 ARGYPRWRLGRRYDY A2A-19 24GFTFSGY 113 SGSGAS 202 ARGYKRWRLGRRYDY A2A-20 25 GFAFSNY 114 YPKSGS 203LYGYDLHWYFDV A2A-21 26 GGSISSGGY 115 NPNSGN 204 DEVAAAGLFDY A2A-22 27GYTFTEY 116 HPSSGS 205 HEVEYYGPSSSWFAY A2A-23 28 GFTFSTY 117 SGSGGS 206ARGKWRWRLGRRYDY A2A-24 29 GFNIGNT 118 NPNYGT 207 DYGSSSFDY A2A-25 30GFTFGNY 119 DPANGD 208 EGDNSNYYAMDY A2A-26 31 GFTFSTY 120 SGSAGS 209ARGHWRWRLGRRYDY A2A-27 32 GFTFSSY 121 SGSGGS 210 ARGYWRWRLWRRYDY A2A-2833 GFTFSSQ 122 SGSGVS 211 ARGRWRWRLGRRYDY A2A-29 34 GYSFTGY 123 YPGSGN212 EDDYGWYFGV A2A-30 35 GYRLTGY 124 DPASGD 213 HEDPIYYGNYVFAY A2A-31 36GYLFTDY 125 YPGTG 214 LYYGSSWERYFDV A2A-32 37 GFTFIDY 126 NPNYGT 215QGSNYGGYFDV A2A-33 38 GFPFSSY 127 SGSGGR 216 ARGYWRWRLGRRADY A2A-34 39GFNFNTY 128 YPGNSD 217 VIYYYGSSDYTLDY A2A-35 40 GFTFSTY 129 SGSGGS 218ARGKWRWRLGRRYDY A2A-36 41 GFNIGNT 130 NPNYGT 219 DYGSSSFDY A2A-37 42GYTFTSY 131 NHDGSN 220 SMITRFAY A2A-38 43 GFSLTSY 132 DPETDD 221YYYGSSAFAY A2A-39 44 GFTFSNY 133 NPNNGG 222 AYYSNYGVMYF A2A-40 45GFNFRSY 134 SGGGGS 223 ARGGWRWRLGRRYDY A2A-41 46 GFSLSIY 135 SPGSGS 224PYYYGSSRYYAMDY A2A-42 47 GYTFTSY 136 SSGGDS 225 DYYGSSWHFDV A2A-43 48GFTFSSY 137 SDGGSY 226 YIWYYGSSWSWYFDA A2A-44 49 GFTFSAY 138 GTAGD 227GYNWIFDL A2A-45 50 GYSFTGY 139 LPGSGG 228 GNYDAMDY A2A-46 51 GGYISSSN140 EQDGSE 229 GEYSRLWYFDL A2A-47 52 GTFTDY 141 LPGSGG 230 PYDYDFDYA2A-48 53 GYTFTSS 142 YPRDGS 231 TVVADWYFDV A2A-49 54 GYTFNDD 143 NPNNGA232 KGDGGSYAAMDY A2A-50 55 GYSFTGY 144 YPKDGS 233 TVVADWYFDV A2A-51 56GYTFNDY 145 NPNNGA 234 NYGSSYYALDY A2A-52 57 GYTFNDY 146 NPNNGG 235QGSNYGGYFDV A2A-53 58 GFNIIDD 147 TDTGEP 236 DYIYAMDY A2A-54 59 GYTFTDY148 DPANGD 237 GDYGSSYAMDY A2A-55 60 GYEFSSS 149 YPGTGN 238 YYYGSSAFAYA2A-56 61 GFTFSSY 150 DPGTGG 239 IYYDYSAMDY A2A-57 62 GFIFSDF 151 DPEDG240 DYYGSSYLDY A2A-58 63 GFNIKDY 152 NPNNGG 241 DYYGSFHRRWYFDV A2A-59 64GYTFTDY 153 NINNGG 242 DYHGSSFYWYFDV A2A-60 65 GYTFTEY 154 NFDGSS 243YYDSSYYAMDY A2A-61 66 GFTFSTY 155 YPGDTD 244 GIAVAGTFDY A2A-62 67GYTFTNY 156 NPNNGG 245 HALLWYYYAMDY A2A-63 68 GFTFSDH 157 NPNSGI 246VSYSGSLHY A2A-64 69 GFTFDDY 158 NTNTGN 247 SNWNYFDY A2A-65 70 GSAFSAS159 DPDNGD 248 PRDSGPSFAS A2A-66 71 GFTFSSY 160 YPKDGS 249SRGYYYGSSYGYYDV A2A-67 72 GHTITSY 161 LPGSGT 250 NWGFAY A2A-68 73GYTFSGY 162 DPSDSF 251 DYGSSYEFTY A2A-69 74 GGYISSSN 163 KTKTDGGT 252GYSGSVDY A2A-70 75 GSNIKDY 164 SDGGS 253 DATGTFAY A2A-71 76 GGSISSSN 165YHSGS 254 EVVSGMIGTVFDY A2A-72 77 GFTISTY 166 GTAGD 255 GYNWIFDY A2A-7378 GFTVSTY 167 GTAGD 256 GYNWIFDF A2A-74 79 GFTFTTY 168 GTAGD 257GYNWIFDF A2A-75 80 GGSISSSN 169 YHSGN 258 EVVSGMIGTIFDY A2A-76 81GFTFSSY 170 GTAGD 259 GYNWIFDF A2A-77 82 GGSISSSN 171 YHSGN 260EVVSGMIGTIFDY A2A-78 83 GFTFSAY 172 GTAGD 261 GYNWVFDL A2A-79 84 GFTFDDY173 TWNGDR 262 DGLTGIFDY A2A-80 85 GFTISTY 174 GTAGD 263 GYNWIFDY A2A-8186 GGSISSSN 175 YHSGS 264 EVVSGLYGTIFDY A2A-82 87 GYSITSGY 176 SYGGS 265DYDYFDY A2A-83 88 GYAFSSY 177 YPGDGD 266 GAY A2A-84 89 GYTFTEY 178SGGGSY 267 PNYSGSSPFAY A2A-85 90 GFSLTAY 179 WTGGG 268 SRGYYYGSSYGYFDVA2A-86 91 GYSITSD 180 NYSGS 269 KLDWDGYFDV A2A-87 92 GFNIKNT 181 DPANGN270 GSPYGYDGHYVMDY A2A-88 93 GFTFRTY 182 SAEGSN 271 DGRGSLPRPKGGFIGALSFHWPFG RWLGGSYGTYDS SEDSGGAFDI A2A-89 94 GFTFNNY 183 SYGGSD 272DGRGSLPRPKGG FIGDLSFHWPFG RWLGKSYGTYDS SEDSGGAFDI

TABLE 16 Variable Light Chain CDRs A2AR  SEQ SEQ SEQ Variant ID NOCDR1 Sequence ID NO CDR2 Sequence ID NO CDR3 Sequence A2A-1 273RSSQSLVYSDGNTYLN 362 KVSNRDS 451 MQGTHWPRT A2A-2 274 KASQDIDDDMN 363EATTLVP 452 LQHDNFPMYT A2A-3 275 KSSQSVLYSSNNKNYLA 364 WASTRES 453QQYYSTPYT A2A-4 276 RASQSVSSNLA 365 GASTRAT 454 QQYYSTPLT A2A-5 277KASQDIDDDMN 366 EATTLVP 455 LQHDNFPWT A2A-6 278 RASQGISSWLA 367 AASSLQS456 QQTNSFPRT A2A-7 279 KASQDVDDDMN 368 EATTLVP 457 LQHDNFPWT A2A-8 280KASQNVGTNVA 369 SASYRYS 458 QRFNNYPLT A2A-9 281 KASQNVGSSVA 370 STSYRYS459 QQYNSYPLT A2A-10 282 RASQSISDYLH 371 YASQSIS 460 QNGHSFPLT A2A-11283 KASRNVGTNVA 372 SASYRYS 461 QQYNSYPLT A2A-12 284 RASQSISDYLH 373YASQSIS 462 QNGHSFPHT A2A-13 285 KASQNVGTNVA 374 SASYRYS 463 QQYNIYPLTA2A-14 286 RASQSISNYLH 375 YASQSIS 464 QNGHSFPLT A2A-15 287 RASQSIGRYLN376 AASSLHS 465 QQSYVTPWT A2A-16 288 RASQSIGTYLN 377 GASTLHS 466QQSYSAPWT A2A-17 289 KASQSVRNDVV 378 RGNTLRP 467 QQYYGIPLT A2A-18 290RASQSVTTYLN 379 SASSLQS 468 QQTYATPWT A2A-19 291 RASQSISDYLN 380 TASTLQS469 EQSYSTPWT A2A-20 292 KASHSVDYDGDNYMN 381 WASTRLT 470 LQHIEYPFTA2A-21 293 KSSQSVLYSSNNKNYFA 382 DAPNRAT 471 QQGYTTPYT A2A-22 294RASQDIGRSLS 383 DASRFIS 472 QWSNSWPYT A2A-23 295 RASQSIGRYLN 384 AASSLHS473 QQSYVTPWT A2A-24 296 KASQSVRNDVV 385 RGNTLRP 474 QQYYGIPLT A2A-25297 KASQSVDYDGDSYMN 386 RANRLVD 475 QNGHSFPLT A2A-26 298 RASQTISRYLN 387SASTLQS 476 QQSYSTPHT A2A-27 299 RASQSIGSYLN 388 GASNLQS 477 QQGYSAPRTA2A-28 300 RASRSISSYLN 389 AASSLPS 478 QQSYSTPRT A2A-29 301 KVSQDVRTAVA390 DTSYLAS 479 QQSYSWSLT A2A-30 302 GGGNDIGSSMY 391 WMSNLAS 480QQYSTYPFA A2A-31 303 RASQSISDYLN 392 GASPRES 481 QQDNIWPYT A2A-32 304GGGNDIGSSMY 393 DASRFIS 482 QQSNEDPPFT A2A-33 305 RASESVDSFGNNFMN 394HTSRLNS 483 QQNNEVPRT A2A-34 306 RASSSVTYIH 395 AVSRLDS 484 HQSNEDPYTA2A-35 307 RASQSIGRYLN 396 AASSLHS 485 QQSYVTPWT A2A-36 308 KASQSVRNDVV397 RGNTLRP 486 QQYYGIPLT A2A-37 309 KASHSVDYDGDNYMN 398 DASRFIS 487LRYASYRT A2A-38 310 RASESVNSYGNSFMH 399 DASRFIS 488 LQYGESPLT A2A-39 311RSSKSLLHSSGNAYVY 400 YTSKPNS 489 QHHYGIPLT A2A-40 312 RASQSIGTYLN 401AASSLES 490 QQTYNTPWT A2A-41 313 RASSRVSSSYLY 402 ATYSLDY 491 LQHGERPLTA2A-42 314 GASQSIGTIIH 403 DTSYLAS 492 QQGNTRPWT A2A-43 315 RASENIYVPLN404 DASRFIS 493 QQYNSFPLYT A2A-44 316 RASQSVSSSYLA 405 GASSRAT 494QQYGSSPIT A2A-45 317 KSSQSLLYSGEKTYPY 406 WASTRLT 495 QQSNEDSWT A2A-46318 QSSQDIFNYLE 407 TASNLDT 496 QQGYSTPPEIT A2A-47 319 RSTRNILSNMP 408NANTLAE 497 LQHWNYPYM A2A-48 320 RASQDISNNLH 409 EISGWLS 498 QQSNSWSLLTA2A-49 321 SASQSMSNNLH 410 LASNLGY 499 RQNGHSFPLT A2A-50 322 RASQDISNNLH411 WASTRLT 500 QQWSDYPFT A2A-51 323 SASSSLSYMH 412 GASPRES 501RQMSSYPPT A2A-52 324 SASSSVSYMN 413 EISGWLS 502 LRYASYRT A2A-53 325KASQNMGSNVA 414 SASHRSS 503 QQWNYPRIT A2A-54 326 KASQNGGTNVD 415 EISGWLS504 QHYYSWPPT A2A-55 327 RASENIYVPLN 416 LASYRFT 505 QQINGWPYT A2A-56328 KASQNMGSNVA 417 AATRLAD 506 RQHYSSPPT A2A-57 329 KASQNGGTNVD 418VASNQGT 507 QQYYTYPLT A2A-58 330 KASQGVDTNVA 419 SSSIS 508 AQNRELPFTA2A-59 331 KASQDVGTAIT 420 SASKRNT 509 LHHYGTPYT A2A-60 332 KASQDVGTSVA421 PASYRSS 510 QQGSSNPLT A2A-61 333 RASQVIDDDIN 422 LGSNRAP 511HQSYTTPHT A2A-62 334 RASQEISGYLT 423 SASHRSS 512 QQWDNNPYT A2A-63 335RASQSISRYLN 424 KASSLER 513 LQPNSYPWT A2A-64 336 RASQGISSWLD 425 TPFSLQS514 QHYDDLPLT A2A-65 337 KASQNMGSNVA 426 EASTRFS 515 QQYSSYPLR A2A-66338 RASQGILGYLN 427 STSNLLL 516 RQLSSNPLT A2A-67 339 RASESVDNYGISFMS 428DASRFIS 517 QQINSWPLT A2A-68 340 KSSQSLLYSGEKTYPY 429 EASNRYT 518QQWSSYPPIA A2A-69 341 RASQGLRHDLG 430 WASNRES 519 QKYSSTPYT A2A-70 342HASESVSVAGTSLLH 431 AASNRES 520 QHWSSFPLT A2A-71 343 RVSQGISNYLN 432AASSLQS 521 QQSYSTPLT A2A-72 344 RASQSVSSNLA 433 GASSRAT 522 QQYGSSPPTA2A-73 345 RASQSVSSNLA 434 GASSRAT 523 QQYGSSPLT A2A-74 346 RASQSVSSSYLA435 GASSRAT 524 QQYYSTPLT A2A-75 347 RASQSISSYLN 436 AASSLQS 525QQANSFPIT A2A-76 348 RASQSVSSNLA 437 DASNRAT 526 QQYGSSPLT A2A-77 349RASQRISSYLN 438 AASSLQS 527 QQSYSTPLT A2A-78 350 RAIQSVSSSYLA 439GASSRAT 528 QQYGSSPLT A2A-79 351 RASQSVSSYLA 440 GASSRAT 529 QQYGNSYTA2A-80 352 RASQSVSSNLA 441 GASTRAT 530 QQYGSSPPT A2A-81 353 RASQSISSYLN442 AASSLQS 531 QQSYSTPIT A2A-82 354 KASQSVSNDVA 443 YASNRYT 532QQDYRSPLT A2A-83 355 KASQNVGTNVA 444 SASYRYS 533 QQYNSYPLT A2A-84 356SASSSVSYMY 445 DTSNLAS 534 QQWNSNPLT A2A-85 357 RASQSISDYLH 446 YASQSIS535 QNGHSFPLT A2A-86 358 HASQNINVWLN 447 KASNLHT 536 QQGQSYPLT A2A-87359 KASQNVGSNVA 448 SASYRYS 537 QQYNSYPLT A2A-88 360 SGISSNIGNNYVS 449DNNKRASG 538 GTWDTSLSAGV A2A-89 361 SGSSSNIGNHYVS 450 DNTKRPSG 539GTWDASLSTWV

TABLE 17 Variable Heavy Chain Sequences A2AR Variant SEQ ID NO SequenceA2A-1 540 QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYPSGNTYYNPSLKSRVTISVDKSKNQFSLKLNSVT AADTAVYYCARDEGYWGQGTLVTVSSA2A-2 541 EVQLLESGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSSLRSEDTAVYYCARGGPFDYWGQGTMVTVSS A2A-3 542EVQLLESGAEVKKPGASVKASCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARVYSYGFDYWGQGTLVTVSS A2A-4 543AGQLQESGGGLVKPGGSLRPSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDNWAFDLWGQGTLVTVSS A2A-5 544GGALVQSGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQATGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLHLQMNSLRDEDTAVYYCARTWYSSSPFDYWGQGTLVTVSS A2A-6 545EVQLLESGGGLVKPGGSPRLSCAASGFTFSNYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVNGRFTISRDYAKNSLYLQMNSLRAEDTAVCYCARDSGSYYDWFDPWGQGTLVTVSS A2A-7 546QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRRAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYSNYFDYWGQGTLVTVSS A2A-8 547EVQLQQPGPGLVKPSLSLSHTCSVTGYSITSGYYWNWIRQFPGKKLEWMGYISYDGSNNYNPSLKNRTSITRDTSKNQFFLKLSSVTTDDTATYYCARVHHYYGSSYFDYWGQGTTLTVSS A2A-9 548EVQLQQSGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGKKLEWMGYIRYDGSNNYNPSLKNRISITRDTSKNQFFLKLNSVTTDDTATYYCARVHHYYGSSYFDYWGQGTTLTVSS A2A-10 549EVQLQQSGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYISYDGSNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCARDPYYYGSSYFDYWGQGTTLTVSS A2A-11 550EVKLVESEGGLVQPGSSMKLSCTASGFTFSDYYMAWVRQVPEKGLEWVANINYDGSSTYYLDSLKSRFTISRDNAKNILYLQMSSLKSEDIATYYCAREYYYGSSSFAYWGQGTTLTVSS A2A-12 551EVNPVESEGGLVQPGSSMKLSCTASGFTFNDYYMAWVRQVPEKGLEWVANINYDGSSTYYLDSLKSRFIISRDNAKNILYLQMSSLKSEDTATYYCAREYYYGSSSFAYWGQGTLVTVSA A2A-13 552GGEVVESGGGLVKPGGSLKLSCAASGFTFSDFGMHWVRQAPEKGLEWVAYISSGSSTIYYADTVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCARREFAYWGQGTLVTVSA A2A-14 553EVKLEESGGGLVKPGGSLKLSCAVSGFTFSDYGMHWVRQAPEKGLEWVAYISSGSGTIYYEDTVKGRFTISRDNAKNTLFLQMTSLRSEGTAIYYCARPNYHGSSPFAYWGQGTLVTVSA A2A-15 554EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYGMSWVRQAPGKGLEWVSGISGSGGSTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAARGKWRWRLGRRYDYWGQGTLVTVSS A2A-16 555EVQLVESGGGLVKPGGSLRLSCAASGFTFNNYAMNWVRQAPGKGLEWVSSISGSGGDTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAARGYWRWRLLRRYDYWGQGTLVTVSS A2A-17 556EVQLVESGGGLVKPGGSLRLSCAASGFNIGNTYMHWFRQAPGKGLEWVGVINPNYGTTRYNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDYGSSSFDYWGQGTLVTVSS A2A-18 557EVQLVESGGGLVKPGGSLRLSCAASGFSFSGYAMSWVRQAPGKGLEWVSVISGSGGSTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAARGYPRWRLGRRYDYWGQGTLVTVSS A2A-19 558EVQLVESGGGLVKPGGSLRLSCAASGFTFSGYAMNWVRQAPGKGLEWVSTISGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAARGYKRWRLGRRYDYWGQGTLVTVSS A2A-20 559EVQLVESGGGLVKPGGSLRLSCAASGFAFSNYWMNWVRQAPGKGLEWVGWFYPKSGSIKYNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTGLYGYDLHWYFDVWGQGTLVTVSS A2A-21 560QVQLVQSGAEVKKPGASVKVSCKASGGSISSGGYYWNWVRQATGQGLEWMGWMNPNSGNRGSAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDEVAAAGLFDYWGQGTLVTVSS A2A-22 561EVQLVESGGGLVKPGGSLRLSCAASGYTFTEYITHWVRQAPGKGLEWVGMIHPSSGSISYNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHEVEYYGPSSSWFAYWGQGTLVTVSS A2A-23 562EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYGMSWVRQAPGKGLEWVSGISGSGGSTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAARGKWRWRLGRRYDYWGQGTLVTVSS A2A-24 563EVQLVESGGGLVKPGGSLRLSCAASGFNIGNTYMHWFRQAPGKGLEWVGVINPNYGTTRYNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDYGSSSFDYWGQGTLVTVSS A2A-25 564EVQLVESGGGLVKPGGSLRLSCAASGFTFGNYWMNWVRQAPGKGLEWVGRIDPANGDTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGDNSNYYAMDYWGQGTLVTVSS A2A-26 565EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMGWVRQAPGKGLEWVSGISGSAGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAARGHWRWRLGRRYDYWGQGTLVTVSS A2A-27 566EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMAWVRQAPGKGLEWVSAISGSGGSTYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAARGYWRWRLWRRYDYWGQGTLVTVSS A2A-28 567EVQLVESGGGLVKPGGSLRLSCAASGFTFSSQAMSWVRQAPGKGLEWVSSISGSGVSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAARGRWRWRLGRRYDYWGQGTLVTVSS A2A-29 568EVQLVESGGGLVKPGGSLRLSCAASGYSFTGYDISWVRQAPGKGLEWVARIYPGSGNTYYDDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREDDYGWYFGVWGQGTLVTVSS A2A-30 569EVQLVESGGGLVKPGGSLRLSCAASGYRLTGYWIEWVRQAPGKGLEWVGRIDPASGDTTYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHEDPIYYGNYVFAYWGQGTLVTVSS A2A-31 570EVQLVESGGGLVKPGGSLRLSCAASGYLFTDYNMNWVRQAPGKGLEWVGWIYPGTGNTYNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTALYYGSSWERYFDVWGQGTLVTVSS A2A-32 571EVQLVESGGGLVKPGGSLRLSCAASGFTFIDYGMHWVRQAPGKGLEWVGVINPNYGTTRYNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQGSNYGGYFDVWGQGTLVTVSS A2A-33 572EVQLVESGGGLVKPGGSLRLSCAASGFPFSSYAMTWVRQAPGKGLEWVSGISGSGGRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAARGYWRWRLGRRADYWGQGTLVTVSS A2A-34 573EVQLVESGGGLVKPGGSLRLSCAASGFNFNTYAMNWVRQAPGKGLEWVGVIYPGNSDTTYNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTGVIYYYGSSDYTLDYWGQGTLVTVSS A2A-35 574EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYGMSWVRQAPGKGLEWVSGISGSGGSTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAARGKWRWRLGRRYDYWGQGTLVTVSS A2A-36 575EVQLVESGGGLVKPGGSLRLSCAASGFNIGNTYMHWFRQAPGKGLEWVGVINPNYGTTRYNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDYGSSSFDYWGQGTLVTVSS A2A-37 576EVQLVESGGGLVKPGGSLRLSCAASGYTFTSYWVHWVRQAPGKGLEWVANINHDGSNTYYLDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSMITRFAYWGQGTLVTVSS A2A-38 577EVQLVESGGGLVKPGGSLRLSCAASGFSLTSYNIDWVRQAPGKGLEWVGGVDPETDDTAYNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCANYYYGSSAFAYWGQGTLVTVSS A2A-39 578EVQLVESGGGLVKPGGSLRLSCAASGFTFSNYYMSWVRQAPGKGLEWVGDINPNNGGTTYNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTKAYYSNYGVMYFWGQGTLVTVSS A2A-40 579EVQLVESGGGLVKPGGSLRLSCAASGFNFRSYAMSWVRQAPGKGLEWVSVISGGGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAARGGWRWRLGRRYDYWGQGTLVTVSS A2A-41 580EVQLVESGGGLVKPGGSLRLSCAASGFSLSIYGISWVRQAPGKGLEWVGDISPGSGSTNYNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGPYYYGSSRYYAMDYWGQGTLVTVSS A2A-42 581EVQLVESGGGLVKPGGSLRLSCAASGYTFTSYNINWVRQAPGKGLEWVATISSGGDSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCERDYYGSSWHFDVWGQGTLVTVSS A2A-43 582EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMYWVRQAPGKGLEWVASISDGGSYTYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYIWYYGSSWSWYFDAWGQGTLVTVSS A2A-44 583EVQLVESGGGVVQPGRSLRLSCAASGFTFSAYDIYWVRQPTGKGLEWVSAIGTAGDTYYPGSVKGRFIISRESAKNSVYLQMNSLRAGDTAVYYCAVGYNWIFDLWGQGTLVTVSS A2A-45 584EVQLVESGGGLVKPGGSLRLSCAASGYSFTGYDISWFRQAPGKGLEWVGEILPGSGGTNYNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTSGNYDAMDYWGQGTLVTVSS A2A-46 585QVQLVQSGAEVKKPGASVKVSCKASGGYISSSNWWSWVRQATGQGLEWMANIEQDGSEKNYVQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGEYSRLWYFDLWGQGTLVTVSS A2A-47 586EVQLVESGGGLVKPGGSLRLSCAASGTFTDYYMKWVRQAPGKGLEWVGEILPGSGGTNYNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARPYDYDFDYWGQGTLVTVSS A2A-48 587EVQLVESGGGLVKPGGSLRLSCAASGYTFTSSWMHWARQAPGKGLEWVGWLYPRDGSTEYNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCITTVVADWYFDVWGQGTLVTVSS A2A-49 588EVQLVESGGGLVKPGGSLRLSCAASGYTFNDDYTNWVRQAPGKGLEWVGNINPNNGAMIYNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARKGDGGSYAAMDYWGQGTLVTVSS A2A-50 589EVQLVESGGGLVKPGGSLRLSCAASGYSFTGYDISWVRQAPGKGLEWVGWIYPKDGSTKYNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCITTVVADWYFDVWGQGTLVTVSS A2A-51 590EVQLVESGGGLVKPGGSLRLSCAASGYTFNDYYINWVRQAPGKGLEWVGDINPNNGANIYNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNYGSSYYALDYWGQGTLVTVSS A2A-52 591EVQLVESGGGLVKPGGSLRLSCAASGYTFNDYYINWVRQAPGKGLEWVGDINPNNGGTTYNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQGSNYGGYFDVWGQGTLVTVSS A2A-53 592EVQLVESGGGLVKPGGSLRLSCAASGFNIIDDYMHWVRQAPGKGLEWVGMITDTGEPTDADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVYDYIYAMDYWGQGTLVTVSS A2A-54 593EVQLVESGGGLVKPGGSLRLSCAASGYTFTDYDMYWVRQAPGKGLEWVGRIDPANGDTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGDYGSSYAMDYWGQGTLVTVSS A2A-55 594EVQLVESGGGLVKPGGSLRLSCAASGYEFSSSWMNWVRQAPGKGLEWVGWIYPGTGNTNYNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCANYYYGSSAFAYWGQGTLVTVSS A2A-56 595EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGLPWVRQAPGKGLEWVGAIDPGTGGTASNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIYYDYSAMDYWGQGTLVTVSS A2A-57 596EVQLVESGGGLVKPGGSLRLSCAASGFIFSDFYMAWVRQAPGKGLEWVGRIDPEDGDEHADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDYYGSSYLDYWGQGTLVTVSS A2A-58 597EVQLVESGGGLVKPGGSLRLSCAASGFNIKDYYMHWVRQAPGKGLEWVGDINPNNGGTTYNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDYYGSFHRRWYFDVWGQGTLVTVSS A2A-59 598EVQLVESGGGLVKPGGSLRLSCAASGYTFTDYNIDWVRQAPGKGLEWVGDININNGGTTYNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDYHGSSFYWYFDVWGQGTLVTVSS A2A-60 599EVQLVESGGGLVKPGGSLRLSCAASGYTFTEYITHWVRQAPGKGLEWVANINFDGSSTYYLDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYDSSYYAMDYWGQGTLVTVSS A2A-61 600QVQLVQSGAEVKKPGASVKVSCKASGFTFSTYIMSWVRQATGQGLEWMGHYPGDTDTRYSQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGIAVAGTFDYWGQGTLVTVSS A2A-62 601EVQLVESGGGLVKPGGSLRLSCAASGYTFTNYLIEWVRQAPGKGLEWVGDINPNNGGTYYNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRHALLWYYYAMDYWGQGTLVTVSS A2A-63 602QVQLVQSGAEVKKPGASVKVSCKASGFTFSDHYMTWVRQATGQGLEWMGWMNPNSGITGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARVSYSGSLHYWGQGTLVTVSS A2A-64 603QVQLVQSGAEVKKPGASVKVSCKASGFTFDDYAMHWVRQATGQGLEWMGVINTNTGNPTYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSNWNYFDYWGQGTLVTVSS A2A-65 604EVQLVESGGGLVKPGGSLRLSCAASGSAFSASWMNLVRQAPGKGLEWVGWVDPDNGDTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSKPRDSGPSFASWGQGTLVTVSS A2A-66 605EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMYWVRQAPGKGLEWVGWIYPKDGSTKYNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSRGYYYGSSYGYYDVWGQGTLVTVSS A2A-67 606EVQLVESGGGLVKPGGSLRLSCAASGHTITSYGINWVRQAPGKGLEWVGEILPGSGTSDYNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATNWGFAYWGQGTLVTVSS A2A-68 607EVQLVESGGGLVKPGGSLRLSCAASGYTFSGYTMHWVRQAPGKGLEWVGEIDPSDSFANYNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDYGSSYEFTYWGQGTLVTVSS A2A-69 608QVQLVQSGAEVKKPGASVKVSCKASGGYISSSNWWSWVRQATGQGLEWMGRIKTKTDGGTIDYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKGYSGSVDYWGQGTLVTVSS A2A-70 609EVQLVESGGGLVKPGGSLRLSCAASGSNIKDYYIHWVRQAPGKGLEWVATISDGGSYIFDDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDATGTFAYWGQGTLVTVSS A2A-71 610QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGRGLEWIGEIYHSGSTNYNPSLKSRVTISVDKPKNQFSLKLSSVTAADTAVYYCAREVVSGMIGTVFDYWGQGTLVTVSS A2A-72 611QVQLVQSGGGLVQPGGSLRLSCAVSGFTISTYDIYWVRQATGKGLEWVSAIGTAGDTYYPDSVRGRFTISREDARNSLYLQMNSLRTGDTAVYYCATGYNWIFDYWGQGTLVTVSS A2A-73 612QVQLVQSGGGLVQPGGSLRLSCAASGFTVSTYDIYWVRQTTGKGLELVSAIGTAGDTYYPDSVKGRFTISRENARNSLYLQMNSLRAGDTAVYYCAVGYNWIFDFWGQGTLVTVSS A2A-74 613EVQLVESGGGLVQPGGSLRLSCAASGFTFTTYDMYWVRQTTGKGLEWVSAIGTAGDTYYPDSVKGRFTISRESAKNSLYLQMNSLRAGDTAVYYCTVGYNWIFDFWGHGTLVTVSS A2A-75 614QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGNTNYNPSLKSRVTMSVDKSKNQFSLNLHSVTAADTAVYYCAREVVSGMIGTIFDYWGQGTLVTVSS A2A-76 615EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMYWVRQPTGKGLEWVSAIGTAGDTYYSGSVKGRFTISRESAKNSLYLQMNSLRAGDTAVYYCAVGYNWIFDFWGQGTLVTVSS A2A-77 616QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGNTNYNPSLKSRVTMSVDKSKNQFSLNLHSVTAADTAVYYCAREVVSGMIGTIFDYWGQGTLVTVSS A2A-78 617EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYDIYWVRQPTGKGLEWVSAIGTAGDTYYPGSVKGRFIISRESAKNSVYLQMNSLRAGDTAVYYCAVGYNWVFDLWGQGTLVTVSS A2A-79 618QVQLQESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQVPGKGLEWVSGITWNGDRSGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCVRDGLTGIFDYWGQGTLVTVSS A2A-80 619QVQLVQSGGGLVKPGGSLRLSCAASGFTISTYDIYWVRQATGKGLEWVSAIGTAGDTYYPGSVKGRFTISRENAKNSLYLQMNSLRAGDTAVYYCASGYNWIFDYWGQGTLVTVSS A2A-81 620QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLGSVTAADTAVYYCAREVVSGLYGTIFDYWGQGTLVTVSS A2A-82 621EVQLQQSGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYISYGGSNDYNPSLKNRISITRDSSKNQFFLKLNSVTTEDTATYYCARDYDYFDYWGQGTTLTVSS A2A-83 622EVQRVQSGAELVKPGASVKISCKASGYAFSSYWMNWVKQRPGKGLEWIGQIYPGDGDTNYNGKFEGKATLTADKSSSTAYMQL TSLTSDDSAVYYCARGAYWGQGTTLTVSSA2A-84 623 EVQLVESGGGLVKPGGSLRLSCAASGYTFTEYITHWVRQAPGKGLEWVATISGGGSYTNFPDSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCARPNYSGSSPFAYWGQGTLVTVSA A2A-85 624EVQLQQSGPGLVAPSQSLSITCTVSGFSLTAYAISWVRQPPGKGLEWLGVIWTGGGTNYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTARYYCARSRGYYYGSSYGYFDVWGTGTTVTVSS A2A-86 625EVQLQESGPGLAKPSQTLPLTCSVIGYSITSDYWNWIRKFPGNKLEYMGYINYSGSTYYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCTRKLDWDGYFDVWGTGTTVTVSS A2A-87 626EVQLQQSEAELVRPGAPVKLSCTASGFNIKNTYMHWVKQRPEQGLEWIGRIDPANGNTKYAPKFQGKATITADTSSNTAYLQLSSLASEDSAVYFCARGSPYGYDGHYVMDYWGQGTSVTVSS A2A-88 627EVQLVESGGGLVKPGGSLRLSCAASGFTFRTYGMHWVRQAPGKGLEWVAVISAEGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGRGSLPRPKGGFIGALSFHWPFGRWLG GSYGTYDSSEDSGGAFDIWGQGTLVTVSSA2A-89 628 QVQLVESGGGVVQPGRSLRLSCAASGFTFNNYGMHWVRQAPGKGLEWVAVISYGGSDKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGRGSLPRPKGGFIGDLSFHWPFGRWLGKSYGTYDSSEDSGGAFDIWGQGTLVTVSS

TABLE 18 Variable Light Chain A2AR Variant SEQ ID NO Sequence A2A-1 629ELVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEA EDVGVYYCMQGTHWPRTFGQGTKVDIKA2A-2 630 ELTLTQSPAFMSATPGDKVNISCKASQDIDDDMNWYQQKPGEAAIFIIQEATTLVPGIPPRFSGSGYGTDFTLTINNIESEDAAYY FCLQHDNFPMYTFGQGTKLEIKA2A-3 631 ELVLTQSPDSLAVSLGERATFNCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPYTFGQGTKVDIK A2A-4 632ELTLTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQAEDVAV YYCQQYYSTPLTFGGGTKVEIK A2A-5633 ELTLTQSPAFMSATPGDKVNISCKASQDIDDDMNWYQQKPGEAAIFIIQEATTLVPGIPPRFSGSGYGTDFTLTINNIESEDAAYY FCLQHDNFPWTFGQGTKVDTKA2A-6 634 ELQMTQSPSSVSASVGDKVTITCRASQGISSWLAWYQQKPGKGPKLLIYAASSLQSGVPSRFSGSGSGTDFTPTISSLQPEDFAT YYCQQTNSFPRTLGQGTKLEIKA2A-7 635 ELTLTQSPAFMSATPGDKVNISCKASQDVDDDMNWYQQKPGEAAIFIIQEATTLVPGIPPRFSGSGYGTDFTLTINNIESEDAAY YFCLQHDNFPWTFGQGTRLEIKA2A-8 636 DIVMTQAQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTVTNVQSED LAEYFCQRFNNYPLTFGAGTKLEIKA2A-9 637 DIVMTQSQKFMSTSVGDRVSATCKASQNVGSSVAWFQQKPGQSPKALIYSTSYRYSGVPDRFTGSGSGTDFTLTISNVQSEDL AEYFCQQYNSYPLTFGAGTKLEIKA2A-10 638 DIVMTQAPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYY CQNGHSFPLTFGAGTKLEIKA2A-11 639 DIVMTQSQKFMSTSVGDRVSVTCKASRNVGTNVAWYQQKLGQSPKTLIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDL AEYFCQQYNSYPLTFGAGTKLEIKA2A-12 640 DIQMTQTPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYY CQNGHSFPHTLGSGTKLEIKA2A-13 641 DIQMIQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTIGNVQSEDL VEYFCQQYNIYPLTFGAGTKLELKA2A-14 642 DIVMTQSPATLSVTPGDSVSLSCRASQSISNYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYY CQNGHSFPLTFGGGTKLELKA2A-15 643 DIQMTQSPSSLSASVGDRVTITCRASQSIGRYLNWYQQKPGKAPKLLIYAASSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQSYVTPWTFGGGTKLEIKA2A-16 644 DIQMTQSPSSLSASVGDRVTITCRASQSIGTYLNWYQQKPGKAPKLLIYGASTLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQSYSAPWTFGGGTKVEIKA2A-17 645 DIQMTQSPSSLSASVGDRVTITCKASQSVRNDVVWYQQKPGKAPKLLIYRGNTLRPGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQYYGIPLTFGQGTKLEIKA2A-18 646 DIQMTQSPSSLSASVGDRVTITCRASQSVTTYLNWYQQKPGKAPKLLIYSASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQTYATPWTFGGGTKVEIKA2A-19 647 DIQMTQSPSSLSASVGDRVTITCRASQSISDYLNWYQQKPGKAPKLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCEQSYSTPWTFGGGTKLEIKA2A-20 648 DIQMTQSPSSLSASVGDRVTITCKASHSVDYDGDNYMNWYQQKPGKAPKLLIYWASTRLTGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCLQHIEYPFTFGQGTKLEIKA2A-21 649 DIQMTQSPSSLSASVGDRVTITCKSSQSVLYSSNNKNYFAWYQQKPGKAPKLLIYDAPNRATGVPSRFSGSGSGTDFTLTISSLQ PEDFATYYCQQGYTTPYTFGGGTKVEIKA2A-22 650 DIQMTQSPSSLSASVGDRVTITCRASQDIGRSLSWYQQKPGKAPKLLIYDASRFISGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQWSNSWPYTFGQGTKLEIKA2A-23 651 DIQMTQSPSSLSASVGDRVTITCRASQSIGRYLNWYQQKPGKAPKLLIYAASSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQSYVTPWTFGGGTKLEIKA2A-24 652 DIQMTQSPSSLSASVGDRVTITCKASQSVRNDVVWYQQKPGKAPKLLIYRGNTLRPGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQYYGIPLTFGQGTKLEIKA2A-25 653 DIQMTQSPSSLSASVGDRVTITCKASQSVDYDGDSYMNWYQQKPGKAPKLLIYRANRLVDGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCQNGHSFPLTFGQGTKLEIKA2A-26 654 DIQMTQSPSSLSASVGDRVTITCRASQTISRYLNWYQQKPGKAPKLLIYSASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQSYSTPHTFGGGTKVEIKA2A-27 655 DIQMTQSPSSLSASVGDRVTITCRASQSIGSYLNWYQQKPGKAPKLLIYGASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQGYSAPRTFGGGTKLEIKA2A-28 656 DIQMTQSPSSLSASVGDRVTITCRASRSISSYLNWYQQKPGKAPKLLIYAASSLPSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQSYSTPRTFGGGTKLEIKA2A-29 657 DIQMTQSPSSLSASVGDRVTITCKVSQDVRTAVAWYQQKPGKAPKLLIYDTSYLASGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQSYSWSLTFGQGTKLEIKA2A-30 658 DIQMTQSPSSLSASVGDRVTITCGGGNDIGSSMYWYQQKPGKAPKLLIYWMSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQYSTYPFALGQGTKLEIKA2A-31 659 DIQMTQSPSSLSASVGDRVTITCRASQSISDYLNWYQQKPGKAPKLLIYGASPRESGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQDNIWPYTFGQGTKLEIKA2A-32 660 DIQMTQSPSSLSASVGDRVTITCGGGNDIGSSMYWYQQKPGKAPKLLIYDASRFISGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQSNEDPPFTFGQGTKLEIKA2A-33 661 DIQMTQSPSSLSASVGDRVTITCRASESVDSFGNNFMNWYQQKPGKAPKLLIYHTSRLNSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQNNEVPRTFGQGTKLEIKA2A-34 662 DIQMTQSPSSLSASVGDRVTITCRASSSVTYIHWYQQKPGKAPKLLIYAVSRLDSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CHQSNEDPYTFGQGTKLEIK A2A-35663 DIQMTQSPSSLSASVGDRVTITCRASQSIGRYLNWYQQKPGKAPKLLIYAASSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQSYVTPWTFGGGTKVEIKA2A-36 664 DIQMTQSPSSLSASVGDRVTITCKASQSVRNDVVWYQQKPGKAPKLLIYRGNTLRPGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQYYGIPLTFGQGTKLEIKA2A-37 665 DIQMTQSPSSLSASVGDRVTITCKASHSVDYDGDNYMNWYQQKPGKAPKLLIYDASRFISGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCLRYASYRTFGQGTKLEIKA2A-38 666 DIQMTQSPSSLSASVGDRVTITCRASESVNSYGNSFMHWYQQKPGKAPKLLIYDASRFISGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCLQYGESPLTFGQGTKLEIKA2A-39 667 DIQMTQSPSSLSASVGDRVTITCRSSKSLLHSSGNAYVYWYQQKPGKAPKLLIYYTSKPNSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQHHYGIPLTFGQGTKLEIKA2A-40 668 DIQMTQSPSSLSASVGDRVTITCRASQSIGTYLNWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQTYNTPWTFGGGTKVEIKA2A-41 669 DIQMTQSPSSLSASVGDRVTITCRASSRVSSSYLYWYQQKPGKAPKLLIYATYSLDYGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCLQHGERPLTFGQGTKLEIKA2A-42 670 DIQMTQSPSSLSASVGDRVTITCGASQSIGTIIHWYQQKPGKAPKLLIYDTSYLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQGNTRPWTFGQGTKLEIK A2A-43671 DIQMTQSPSSLSASVGDRVTITCRASENIYVPLNWYQQKPGKAPKLLIYDASRFISGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQYNSFPLYTFGQGTKLEIKA2A-44 672 ELVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAV YYCQQYGSSPITFGQGTKVDIKA2A-45 673 DIQMTQSPSSLSASVGDRVTITCKSSQSLLYSGEKTYPYWYQQKPGKAPKLLIYWASTRLTGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCQQSNEDSWTFGQGTKLEIKA2A-46 674 DIQMTQSPSSLSASVGDRVTITCQSSQDIFNYLEWYQQKPGKAPKLLIYTASNLDTGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQGYSTPPEITFGGGTKVEIKA2A-47 675 DIQMTQSPSSLSASVGDRVTITCRSTRNILSNMPWYQQKPGKAPKLLIYNANTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCLQHWNYPYMFGQGTKLEIKA2A-48 676 DIQMTQSPSSLSASVGDRVTITCRASQDISNNLHWYQQKPGKAPKLLIYEISGWLSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQSNSWSLLTFGQGTKLEIKA2A-49 677 DIQMTQSPSSLSASVGDRVTITCSASQSMSNNLHWYQQKPGKAPKLLIYLASNLGYGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCRQNGHSFPLTFGQGTKLEIKA2A-50 678 DIQMTQSPSSLSASVGDRVTITCRASQDISNNLHWYQQKPGKAPKLLIYWASTRLTGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQWSDYPFTFGQGTKLEIKA2A-51 679 DIQMTQSPSSLSASVGDRVTITCSASSSLSYMHWYQQKPGKAPKLLIYGASPRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CRQMSSYPPTFGQGTKLEIK A2A-52680 DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKLLIYEISGWLSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCLRYASYRTFGQGTKLEIK A2A-53681 DIQMTQSPSSLSASVGDRVTITCKASQNMGSNVAWYQQKPGKAPKLLIYSASHRSSGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQWNYPRITFGQGTKLEIKA2A-54 682 DIQMTQSPSSLSASVGDRVTITCKASQNGGTNVDWYQQKPGKAPKLLIYEISGWLSGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQHYYSWPPTFGQGTKLEIKA2A-55 683 DIQMTQSPSSLSASVGDRVTITCRASENIYVPLNWYQQKPGKAPKLLIYLASYRFTGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQINGWPYTFGQGTKLEIKA2A-56 684 DIQMTQSPSSLSASVGDRVTITCKASQNMGSNVAWYQQKPGKAPKLLIYAATRLADGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCRQHYSSPPTFGQGTKLEIKA2A-57 685 DIQMTQSPSSLSASVGDRVTITCKASQNGGTNVDWYQQKPGKAPKLLIYVASNQGTGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQYYTYPLTFGQGTKLEIKA2A-58 686 DIQMTQSPSSLSASVGDRVTITCKASQGVDTNVAWYQQKPGKAPKLLIYSSSISGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CAQNRELPFTFGQGTKLEIK A2A-59687 DIQMTQSPSSLSASVGDRVTITCKASQDVGTAITWYQQKPGKAPKLLIYSASKRNTGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCLHHYGTPYTFGQGTKLEIKA2A-60 688 DIQMTQSPSSLSTSVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYPASYRSSGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQGSSNPLTFGQGTKLEIKA2A-61 689 DIQMTQSPSSLSASVGDRVTITCRASQVIDDDINWYQQKPGKAPKLLIYLGSNRAPGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCHQSYTTPHTFGGGTKVEIKA2A-62 690 DIQMTQSPSSLSASVGDRVTITCRASQEISGYLTWYQQKPGKAPKLLIYSASHRSSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQWDNNPYTFGQGTKLEIKA2A-63 691 DIQMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPKLLIYKASSLERGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCLQPNSYPWTFGGGTKVEIKA2A-64 692 DIQMTQSPSSLSASVGDRVTITCRASQGISSWLDWYQQKPGKAPKLLIYTPFSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQHYDDLPLTFGGGTKVEIKA2A-65 693 DIQMTQSPSSLSASVGDRVTITCKASQNMGSNVAWYQQKPGKAPKLLIYEASTRFSGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQYSSYPLRFGQGTKLEIKA2A-66 694 DIQMTQSPSSLSASVGDRVTITCRASQGILGYLNWYQQKPGKAPKLLIYSTSNLLLGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCRQLSSNPLTFGQGTKLEIKA2A-67 695 DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMSWYQQKPGKAPKLLIYDASRFISGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQINSWPLTFGQGTKLEIKA2A-68 696 DIQMTQSPSSLSASVGDRVTITCKSSQSLLYSGEKTYPYWYQQKPGKAPKLLIYEASNRYTGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCQQWSSYPPIAFGQGTKLEIKA2A-69 697 DIQMTQSPSSLSASVGDRVTITCRASQGLRHDLGWYQQKPGKAPKLLIYWASNRESGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQKYSSTPYTFGGGTKVEIKA2A-70 698 DIQMTQSPSSLSASVGDRVTITCHASESVSVAGTSLLHWYQQKPGKAPKLLIYAASNRESGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQHWSSFPLTFGQGTKLEIKA2A-71 699 ELQMTQSPSSLSASVGDRVTITCRVSQGISNYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQSYSTPLTFGGGTKVEIKA2A-72 700 ELTLTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVY YCQQYGSSPPTFGQGTRLEIKA2A-73 701 ELTLTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVY YCQQYGSSPLTFGPGTKVDIKA2A-74 702 ELVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISSLQAEDVA VYYCQQYYSTPLTFGGGTKVEIKA2A-75 703 ELVMTQFPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQANSFPITFGQGTRLEIKA2A-76 704 ELVMTQSPATLSVSLGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISRLGPEDFA VYYCQQYGSSPLTFGGGTKVEIKA2A-77 705 ELVMTQSPSSLSASVGDRVTITCRASQRISSYLNWYQQKPGKAPKLLIYAASSLQSRVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQSYSTPLTFGGGTKLETKA2A-78 706 ELTLTQSPATLSLSPGERATLSCRAIQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAV YYCQQYGSSPLTFGGGTRLEIKA2A-79 707 ELTLTQSPATLSVSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVY YCQQYGNSYTFGQGTKVDIK A2A-80708 ELTLTQSPGTLSLSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGTPARFSGSGSGTEFTLTISSLQSEDFAVY YCQQYGSSPPTFGQGTRLEIKA2A-81 709 ELVMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQSYSTPITFGGGTKLEIKA2A-82 710 DIVITQAPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKPGQSPKLLIYYASNRYTGVPDRFSGSGYGTDFTFTISTVQAEDLA VYFCQQDYRSPLTFGAGTKLELKA2A-83 711 DIQMKQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSGDL AEYFCQQYNSYPLTFGAGTKLEIKA2A-84 712 DIVMTQAPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRFLIYDTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAAT YYCQQWNSNPLTFSAGTKLEIKA2A-85 713 DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVEPEDVGVY YCQNGHSFPLTFGAGTKLELKA2A-86 714 DIKITQSPSSLSASLGDTITITCHASQNINVWLNRYQQKPGNIPKLLIYKASNLHTGVPSRFSGSGSGTGFTLTISSLQPEDIATYYC QQGQSYPLTFGAGTKLEIK A2A-87715 DIQMNQSQKFMSTSVGDRVSVTCKASQNVGSNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTPTISNVQSEDL AEYFCQQYNSYPLTFGAGTKLELKA2A-88 716 QSVLTQPPSVSAAPGQKVTISCSGISSNIGNNYVSWYQQLPGTAPKLLIYDNNKRASGIPDRFSGSKSGTSATLGITGLQTGDEAD YYCGTWDTSLSAGVFGGGTKLTVLA2A-89 717 QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNHYVSWYQQLPGTAPKLLIYDNTKRPSGIPDRFSGSKSGTSATLGITGLQTGDEA DYYCGTWDASLSTWVFGGGTKLTVL

Example 23: In Vivo Cell Analysis of A2A-77 and A2A-81

Cell Binding Assay

A2A-77 and A2A-81 were assessed for binding at concentrations titratedfrom 100 nM. Resulting curves are shown in FIG. 33A and results areshown in Table 19. Binding curves are plotted with IgG concentration vs.MFI (mean fluorescence intensity). Both A2A-77 and A2A-81 were highaffinity binders to hA2a receptor.

TABLE 19 A2A-77 A2A-81 Control A2A IC50 6.436 6.813 8.723

A2A Antagonistic cAMP Assay

Immunoglobulins were titrated in triplicate and incubated on cells for 1hour, followed by incubation with 0.5 μM NECA for 30 minutes. Bindingcurves showing relative fluorescent units (RFU) ratio at 665 nm/615 nmversus nM IgG on a log scale are shown in FIG. 33B. Absolute IC50 isshown in Table 20, indicating that A2A-77 and A2A-81 were functionalagonists in vitro.

TABLE 20 A2A-77 A2A-81 IC50 3.53 8.67

Cross Reactivity

A2A-77 and A2A-81 were assayed for cross reactivity with HAL hA2b, hA3and mA2 receptor. Results are depicted in FIG. 33C. Both A2A-77 andA2A-81 showed in vitro specificity.

Primary T-Cell Activation Assay

A primary T-cell activation assay was performed as described above. Datais seen in FIG. 33D and Table 21. T-cell activation was observed withvariants A2A-77 and A2A-81. A2A-81 showed improved activity over A2A-77.

TABLE 21 A2A-77 A2A-81 EC50 5.92 1.71

Example 24: In Vivo Study in Colon Carcinoma Model

Mice with human colon carcinoma (Colo205) were divided into 4 groups.Group 1 was the isotype control, Group 2 mice were treated withAnti-PD1, Group 3 mice were treated with variant A2A-77 and Group 4 micewere treated with variant A2A-81. Tumor volume is measured over 30 days.Results are depicted in FIGS. 34A-34D. Variant A2A-81 regressed tumorsize better than variant A2A-77 or the anti-PD-1 antibody.

Additional studies were performed with mice treated with 10 mg/kgvariant A2A-51 (Group 5), A2A-28 (Group 6), Ab7/PD1TAO15 (Group 7), andAZD4635 (Group 8). Mice were also treated with 20 mg/kg according to theschedule in FIG. 34E. Data is seen in FIGS. 34F-34K.

The data shows that A2A-77 and A2A-51 exhibited ability to reduce tumorvolume and PD1TAO15 exhibited similar results to comparator Anti-PD1antibody. No difference was observed in combination treatments ascompared to single treatment or Anti-PD1 antibody treatment. See FIG.35K.

Tumor infiltrating lymphocytes (TIL) in both the lymphoid and myeloidcompartments were measured in each of the treatment groups. Results aredepicted in FIGS. 35A-35M. TIL CD8+ cells increased more in the grouptreated with the A2A-77 variant than the A2A-81 variant. TIL-M1 tumorassociated macrophages increased more in the A2A-81 variant than theA2A-77 variant.

The cell profile of lysed whole blood (LWB) of peripheral blood wasmeasured in interim and terminal samples. The results are depicted inFIGS. 36A-36C, FIGS. 37A-37G, and FIGS. 38A-38G. Cytokine levels inperipheral blood after T cell activation is depicted in FIG. 39. Resultsof the cytokine levels in terminal serum is depicted in FIGS. 40A-40G.

The cell profile of lysed whole blood (LWB) of peripheral blood wasmeasured in interim and terminal samples. The results are depicted inFIGS. 41A-41C, 42A-42G and 43A-43G. Results of the cytokine levels interminal serum samples is depicted in FIGS. 44A-44G.

Example 25: A2bR Cell Functional cAMP Assay

Cross Reactivity

A2b cross binders were assessed for specificity in HEK293T cells wereassayed for cross reactivity. Results are depicted in FIG. 45 and Table22.

TABLE 22 A2a antagonists wA2a antagonists (1^(st) (narrow screen) downleads) Functional Functional Cross react hA1, PE antagonistic cAMPDiscoverX cAMP A2b, A3 A2A-17, A2A-19 A2A-19 A2A-17, A2A-19 A2A-24,A2A-26, A2A-27, A2A-24, A2A-26, A2A-26, A2A-27 A2A-27 A2A-35, A2A-36,A2A-37 A2A-37 A2A-35, A2A-37 A2A-51, A2A-52, A2A-53 A2A-51, A2A-53A2A-74, A2A-75 A2A-75 A2A-77, A2A-78, A2A-81 A2A-78, A2A-81 A2A-83,A2A-84 A2A-83, A2A-84 A2A-83

FIG. 46 depicts the functional cAMP assay performed on selected A2bantibodies. CHO-K1 cells were incubated with an A2b antibody. Next, thecells were stimulated using NECA. Activation of A2b was monitored basedon 3′-5′-cyclic adenosine monophosphate (cAMP) production in the celllines.

A functional allosteric cAMP assay was performed. Cells werepre-incubated with anti-A2A-17, A2A-19, A2A-26, A2A-27, A2A-35, A2A-36,A2A-83, and A2A-84 at 100 nM, followed by NECA stimulation at a 3×titration from 300 nM. The results are depicted in FIG. 47A.

A functional antagonistic cAMP assay was performed. First, cells werepre-incubated with A2A-17, A2A-19, A2A-26, A2A-27, A2A-35, A2A-36, andA2A-83 at a 3× titration starting at 100 nM. Next NECA stimulation at aconcentration of 10 nM was performed. The results are depicted in FIGS.47B-47C.

An antagonistic cAMP assay at high levels of IgC and low ligand levelswas performed. Cells were pre-incubated with A2A-17, A2A-19, A2A-26,A2A-27, A2A-35, A2A-36, A2A-83, and A2A-72 at a 3× titration starting at1000 nM. Next, NECA stimulation was performed at a 5 nM concentration.The results are depicted in FIG. 47D. A2A-27 showed properties of beingan A2a antagonist that cross links with A2b, while also being an A2bantagonist. Properties of A2b are depicted in Table 23.

TABLE 23 Cross reactive hA1, A2b, A3 HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3A2A-27 GFTFSSY SGSGGS ARGYVVRWR RASQSIG GASNLQS QQGYSAPRT (Alloy)(SEQ ID (SEQ ID LWRRYDY SYLN (SEQ ID (SEQ ID NO: NO: 10) NO: 101)(SEQ ID NO: (SEQ ID NO: 388) 477) 210) NO: 299)

Example 26. IgG1 and IgG4 Reformatted A2AR Antibodies

Antibodies were reformed into either IgG1 or IgG4. The reformattedantibodies were then assayed in primary T cell activation assays, whichmeasures cytokine release. Data is seen in FIGS. 48A-48E. As seen in thedata, after reformatting the leads into IgG4, IgG4s have better T cellactivation activities than IgG1s.

Example 27: In Vivo Study in Colon Carcinoma Model

Mice with human colon carcinoma (HuCD34NCG-Colo205) were divided intotwo sets: Set 1 was divided into eight groups and Set 2 was divided intosix groups. In Set 1, Group 1 was the isotype control, Group 2 mice weretreated with Anti-PD1, Group 3 mice were treated with variant Ab3, Group4 mice were treated with variant Ab4, Group 5 mice were treated withvariant Ab5, Group 6 mice were treated with variant Ab6, Group 7 micewere treated with variant Ab7, Group 8 mice were treated with AZD4635.Each group in Set 1 was given a 10 mg/kg dose on a Q3Dx6 schedule exceptfor group 8, which was given 50 mg/kg on a twice daily schedule. In Set2, Group 1 was the isotype control, Group 2 mice were treated withAnti-PD1, Group 3 mice were treated with Ab4, Group 4 mice were treatedwith Ab4+Anti-PD1, Group 5 mice were treated with AB7, and Group 6 micewere treated with AB4+Ab7. Each group in Set 2 was given a 20 mg/kgtotal dose on a Q3Dx6 schedule. Tumor volume was measured over 30 days.

While preferred embodiments of the present disclosure have been shownand described herein, it will be obvious to those skilled in the artthat such embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the disclosure. It should beunderstood that various alternatives to the embodiments of thedisclosure described herein may be employed in practicing thedisclosure. It is intended that the following claims define the scope ofthe disclosure and that methods and structures within the scope of theseclaims and their equivalents be covered thereby. U.S.

What is claimed is:
 1. A method for activating T cells, comprisingadministering an antibody or antibody fragment comprising a sequence atleast about 90% identical to a sequence set forth in SEQ ID NOs: 6-717.2. The method of claim 1, wherein the antibody or antibody fragmentcomprises an amino acid sequence at least about 95% identical to thatset forth in any one of SEQ ID NOs: 35-44.
 3. The method of claim 1,wherein the antibody or antibody fragment comprises an amino acidsequence as set forth in any one of SEQ ID NOs: 35-44.
 4. The method ofclaim 1, wherein the antibody is a monoclonal antibody, a polyclonalantibody, a bi-specific antibody, a multispecific antibody, a graftedantibody, a human antibody, a humanized antibody, a synthetic antibody,a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv), asingle chain antibody, a Fab fragment, a F(ab′)2 fragment, a Fdfragment, a Fv fragment, a single-domain antibody, an isolatedcomplementarity determining region (CDR), a diabody, a fragmentcomprised of only a single monomeric variable domain, disulfide-linkedFvs (sdFv), an intrabody, an anti-idiotypic (anti-Id) antibody, or abantigen-binding fragments thereof.
 5. The method of claim 1, wherein theantibody or antibody fragment binds to adenosine 2A receptor with aK_(D) of less than about 75 nM.
 6. The method of claim 1, wherein theantibody or antibody fragment binds to adenosine 2A receptor with aK_(D) of less than about 50 nM.
 7. The method of claim 1, wherein theantibody or antibody fragment binds to adenosine 2A receptor with aK_(D) of less than about 25 nM.
 8. (canceled)
 9. The method of claim 1,wherein the antibody or antibody fragment comprises an IC₅₀ of less thanabout 20 nM in a T cell activation assay.
 10. The method of claim 1,wherein the antibody or antibody fragment comprises an IC₅₀ of less thanabout 10 nM in a T cell activation assay.
 11. The method of claim 1,wherein the antibody or antibody fragment comprises an IC₅₀ of less thanabout 7.5 nM in a T cell activation assay.
 12. (canceled)
 13. Anantibody or antibody fragment comprising a sequence at least about 90%identical to a sequence set forth in SEQ ID NOs: 6-717.
 14. The antibodyor antibody fragment of claim 13, wherein the antibody or antibodyfragment comprises an amino acid sequence at least about 95% identicalto that set forth in any one of SEQ ID NOs: 35-44.
 15. The antibody orantibody fragment of claim 13, wherein the antibody or antibody fragmentcomprises an amino acid sequence as set forth in any one of SEQ ID NOs:35-44.
 16. The antibody or antibody fragment of claim 13, wherein theantibody is a monoclonal antibody, a polyclonal antibody, a bi-specificantibody, a multispecific antibody, a grafted antibody, a humanantibody, a humanized antibody, a synthetic antibody, a chimericantibody, a camelized antibody, a single-chain Fvs (scFv), a singlechain antibody, a Fab fragment, a F(ab′)2 fragment, a Fd fragment, a Fvfragment, a single-domain antibody, an isolated complementaritydetermining region (CDR), a diabody, a fragment comprised of only asingle monomeric variable domain, disulfide-linked Fvs (sdFv), anintrabody, an anti-idiotypic (anti-Id) antibody, or ab antigen-bindingfragments thereof.
 17. The antibody or antibody fragment of claim 13,wherein the antibody or antibody fragment binds to adenosine 2A receptorwith a K_(D) of less than about 75 nM.
 18. The antibody or antibodyfragment of claim 13, wherein the antibody or antibody fragment binds toadenosine 2A receptor with a K_(D) of less than about 50 nM.
 19. Theantibody or antibody fragment of claim 13, wherein the antibody orantibody fragment binds to adenosine 2A receptor with a K_(D) of lessthan about 25 nM.
 20. (canceled)
 21. The antibody or antibody fragmentof claim 13, wherein the antibody or antibody fragment comprises an IC₅₀of less than about 20 nM in a T cell activation assay.
 22. The antibodyor antibody fragment of claim 13, wherein the antibody or antibodyfragment comprises an IC₅₀ of less than about 10 nM in a T cellactivation assay.
 23. The antibody or antibody fragment of claim 13,wherein the antibody or antibody fragment comprises an IC₅₀ of less thanabout 7.5 nM in a T cell activation assay.
 24. (canceled)